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- This, of all games, has one where in one of the endings, Isaac picks up a fetus in a jar...and is shown wearing a Dr Fetus Costume.
- Unlocking a treasure chest, only to find another treasure chest inside it. It appears Edmund likes to toy with expectations.
- Or unlocking a treasure chest, finding another treasure chest in it, and then finding another treasure chest in that.
- Number One. The look on Isaac's face as he chooses to pee on everything is just priceless.
- Isaac can wear his mother's brassiere or her menstrual pad as items. Doing so causes the enemies to freeze or back away, respectively. Presumably, they're Squicked out by the sight of it!
- Segmented enemies like Larry Jr. will even fall apart when they try to flee. They are so disgusted that it breaks the laws of physics.
- When you meet Greed in a hidden room, you can immediately leave through the hole you made without having to defeat him. Hi, Greed. Bye, Greed. If you have X-ray glasses, you can even use his room as a shortcut, running past him without ever engaging him.
- The beggar that runs the shell game in the arcade is a jerk. Giving you a giant grin as he takes all your money without giving you the items you want? Seeing the slot machine throw a troll bomb at him is just too hilarious a karma to be true.
- There are times where beggars will unveil a troll bomb right next to themselves.
- In Wrath of the Lamb, getting the Speed Ball item (a syringe with white fluid in it) gives Isaac very large, dilated eyes and a little smile, kinda like this: 0u0. It looks incredibly goofy (though less so once you see the bloody syringe sticking out the back of his head).
- The glitches in this game can sometimes cause hilarious reactions from players. Especially since, given the tone of the game and previous games by Edmund Mcmillen, it's difficult to tell if some of them are actually glitches.
- The Binding of Isaac - Secret Ending Cutscene, an animation made by Alon Dan, who made the cutscenes for Rebirth. In the animation, Isaac befriends and proposes a wedding to a Mulligan and a fly that it spits out, then tells his mom to get off in a dramatic expression.
"WE'RE NOW ENGAGED!! ...and you too. OH NO! IT IS MY WEDDING, NOT YOURS!! YOU ARE NOT INVITED, STUIPID MOM!!"
—Isaac
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