Largest in-game surprise you have experienced?
Let me preface this by repeating that old GM adage: No carefully-planned plot will survive contact with the players. Like many GMs out there, I think I have learned this the hard way. So these days I just set the scene, identify what would happen if the PCs don't intervene, and let things unfold. I find that Dungeon World's Fronts work very well for this, regardless of the actual system. So yeah, as a GM my style is one that allows me to be constantly surprised by what the players think of. It seems to work for me.
With that said, I don't have any super-memorable surprises from any of my players. So instead I will talk about surprises that I like to spring as a GM. When it works, it really works. (See +Rachel Teng's Day 10 #RPGaDAY blog post.) But you also have to be careful, lest you spring a surprise for surprise's sake. That's not really cool. The biggest thing a GM needs to accept about surprises in the game is that the best ones are those that the players could have found out. Since there's a chance to find out, GMs should be alright with them actually finding out. You have to reward cleverness, because that's one of the hallmarks of finding satisfaction as an RPG player.
Anyway. That's all I have to say about that.