Fish and Crystal are the game's two starting characters, depending on how good you are at the game (or your preferences) you will probably be using them a lot.

• Fish B-Skin: Loop with all characters. It's a daunting task, but worth it in my opinion.
  
• Fish C-Skin: Unlock all B-Skins. This is easier said than done.

• Crystal B-Skin: As Crystal, reach the Cursed Crystal Caves (4-1). This one isn't too hard to set up, and you may have done it by accident already.
  
  • At any point before 4-1, find and equip a Cursed Weapon. Cursed Weapons can be found in Cursed Weapon Chests, you can tell them apart by the purple on them, rather than the gray of a normal weapon chest. Cursed Weapons emit purple smoke, and have a purple outline when equipped
    
  • Prior to reaching 4-1, do not: touch the Crown Pedestal in a Crown Vault, take the Last Wish mutation, or loop (how?). Any of these actions will "Cleanse" the weapon, removing the curse.
    
  • Play like normal and get to 4-1. You will know if you did it right.
    
  You don't have to beat the Cursed Crystal Caves, you just have to get to there. The skin should unlock when you get there, as long as you are Crystal.
  
  
• Crystal C-Skin: As Crystal, survive a total of 100 damage over the course of a single run.

Eyes, Plant, Robot, and Steroids vary in unlock toughness. You're probably gonna need to practice some before unlocking them (not with Eyes though, I hope). All of them are put in the order they are unlocked. As a side note, you don't have to beat the level to unlock the character. So even if you die on, say 5-1, you would still unlock Robot.

Eyes - Reach 2-1.
Eyes B-Skin - Reach the Pizza Sewers. Look for a manhole, then shoot at it with some sort of explosive weapon

Plant - If you manage to get through the Sewers alright, you'll be transported to 3-1, the Scrapyard.
Plant B-Skin - Reach the Nuclear Throne in under 10 minutes. This should be a breeze for you speedrunners out there.

Robot - Having played the game for almost three years, I'd like to think I know something about the Crystal Caves.
1.) Any bolt weapon is awesome in Caves. Their range, and generally high damage makes them really good for the spiders.
2.) The Bouncer guns also work great here because of their firerate.
3.) Don't get cocky. If you rush into a crowd of five spiders with a piece of looseleaf paper as your weapon, you will die. Take it slow.
If you take that advice or just take it slow in the Crystal Caves, I garuntee you can get to 5-1 and unlock Robot.
Robot B-Skin - Eat a Hyper Weapon. Boring, I know.

Steriods - Lil' Hunter is your first big endurance test in the game, and is an ass to fight when you're new, here's a bit of advice.
1.) High damage weapons work great against Lil' Hunter, examples being the Assault Slugger or the Super Crossbow. YV is GREAT for speedkills if you want him dead fast.
2.) The Heavy and Auto Crossbows work wonders on Lil' Hunter, with the addition of the Seeker Shotgun. Bolt Marrow is highly reccomended if you have any of these weapons.
3.) The bolts that stick into Lil' Hunter will give away where he's landing. Shoot him with a Crossbow if you want to track him down! (Or you can look for the shadow)
4.) Try to bait him into landing away from your cover, so that he doesn't destroy it.
5.) Rush LH while he spawns IDPD, he can't attack you when he does it.
6.) Stay out of open areas. That siper rifle is dangerous.
Even with these tips Lil' Hunter can still crush you. Be cautious when fighting him and you should have no problem getting to 6-1 and unlocking Steriods.

http://nuclear-throne.wikia.com/wiki/Lil'_Hunter
Steriods B-Skin - Kill the Technomancer. This boss spawns in 6-1 during loops.

Die. When attempting to unlock Melting don't even try playing the game. Make it funny, roll into a Scorpion or something.
Melting B-Skin - Reach the Throne without Rihno Skin or Strong Spirit.

Most of the difficulty from this unlock comes from the RNG involved. The first thing you'll need to find is a Screwdriver. Then you'll need to survive until 3-1, so I'd recommend finding a solid weapon. On 3-1, you're going to need to find a red and gold car, then hit it with the Screwdriver. Going up to the car should allow you to use your interact key on it (mapped to E on keyboards) you don't need to go in immediately though. If you do decide to stay behind for the extra rads, stay away from the car, because it can still be destroyed. After entering the car, you will be transported to Y.V.'s Mansion, fulfilling the unlock requirement.

Y.V's B-Skin - Get a golden weapon for every character, the Golden Revolver does count.

Loop back to the Desert to unlock Rebel. Makes sense I guess?

Rebel B-Skin - Defeat Mom. Just let that sink in for a moment....

To unlock Chicken you have to go to the Jungle. In order to get to the Jungle you have to grab Last Wish during your run, then on 5-1, a plant/flower thingy will spawn in somewhere. Feed it 4 HP and you will be taken to the Jungle. But be careful because the flower can be killed. Gamma Guts makes the Flies very easy to kill, just getting that out there.

Chicken B-Skin - Reach 2-1 on Hard Mode as Chicken. I would highly reccomend a melee weapon like the Golden Wrench for dealing with maggots. Good luck.

To unlock Horror, you first have to kill him. This can be achieved on any level, all you have to do is avoid destroying two rad canisters in a row, and Horror should spawn on the third level. Killing Horror is a bit more difficult, because he destroys projectiles with his main attack. Just wait for a good opportunity to strike and you should do fine.

Horror B-Skin - Kill the Hyper Crystal.

Sit on the Throne.
Rogue B-Skin - Murder the Captain's face in HQ3.

Crowns are slightly trickier.

In order to unlock a crown so you can use it in the character select screen you must either loop or sit on the Throne, you'll then recieve a popup stating that you've unlocked the crown. Same goes for loop crowns. Some added advice, unlocking loop crowns is much easier if you kill the Captain.

These unlocks don't really fit into the rest of the guide, so I've given them their own section.

Skeleton - Skeleton is a bit of a toughie. To play him, you must reach the labs as Melting, and find a Necromancer, you must then die in his Res Circle. From there you can play as Skeleton. Prepare to shoot like ♥♥♥♥, walk slow and get ♥♥♥♥♥♥. You may want to grab the Blood Hammer if you see it, the process becomes a lot easier.

Golden Ballguy/Frog - This is going to be a long one. To play as the Frog you have to get Mom to drop a Rusty Frog Pistol. This can be achieved by hitting her at least once with any golden weapon. You must then insert said Rusty Frog Pistol into a Proto Chest in one of the Crown Vaults. Go back as whoever and open up the Proto Chest. A Golden Frog Pistol should have spawned. Pick it up and progress to the next level. Once you die you can then equip the Golden Frog Pistol as your starter. You will then spawn as Frog.

Cuz - YV's cousin, the character was possibly scrapped, or is in development. We're still in the dark about this. Maybe you need to die in a Res Circle as YV?

Hard Mode - Hard Mode is unlocked by reaching L2 0-1. There's no need to beat Throne 2 you only need to reach the level.