When I first started running Dungeons and Dragons I wanted to be the best Dungeon Master that anyone had ever played under. I read what blogs there were on the subject, scoured forums, and listened to all the Dungeon Masters I knew as they told me their 'secrets.' I hoped that by doing all of this research that I would be able to be as great a Dungeon Master as I imagined all these others were.
After spending nearly two years researching different styles of Dungeon Mastering I discovered what I thought was a hidden truth. The best and most exciting games I had been involved in, and read about, all had Dungeon Masters who seemingly never said the word "No." When the realization hit me I decided that I would never refuse my players; and like a fool I announced my decision to my players. They smiled at me and I had the sinking feeling that I had just fucked up.
The next night the gloves came off. They murdered and tortured their way though the underground kingdom of Goblins. It was a game within the game with my players attempting to find something that would force me to stop saying yes.
In the end, as a group, we discovered that the lines none of us would cross should have been obvious to all of us from the beginning: rape and harm to little kids. Since that time there have been a few other points where I didn't like what was going on and stopped it - like the time one of the players was getting picked on through the game - but in general those are the two lines that none of us are willing to cross.
What about you? What things in your games are the lines in the sand that you won't cross?