This is a very interesting question and one that I had no problem answering until a few weeks ago. I ran a game a few weeks ago for my own Pathfinder game that may make me question that game. I am not sure if this recent game has the same impact as the one I normally think of when asked this question.
The normal answer
I once ran a Shadowrun game that was centered on a haunted house. It was a one shot, ran for about five hours and everyone was blown away by the game. By the handling of the system, the NPC’s, the story and the overall thematic success. It placed the players on the edge of a knife and at the end of the game, after they had succeeded there was that buzz of exhilaration from them all. This game was run in around 1993/94 and I have always held on to this memory as an example of that time I got every single thing just so.
The contender
I have been running a Pathfinder game my way. Running a sand box styled game that investigates the lands of dream through the players eyes. It started by having them killed in the material plane and placed into indentured servitude when they were resurrected in the plane of Dreams by an organisation. The players hated the organisation they were in – some more notably than others. It was that organisation that marked them as desirable candidates on the material plane and the person that murdered them actually worked in the Dream Plane alongside them after their resurrection. Hatred and loathing was rife among the group. I worked toward having them realize that while the groups methods were questionable the overall aim was decent. Some began to come around – others did not.
Then a few weeks back I ran a game where an opposing group attempted to take two of the group into their own and have the rest killed. The players killer on the material plane joined them in battling the intruder. He was stabbed, poisoned and died as the players looked on helpless. The madame of the house arrived (Caby from this post) arrived and was destroyed. Biondello (the member of her organization that had died) and she had spent years working together and she was gutted. Totally destroyed at his loss.
The players as they left this game were all affected by this game. The player that was the most anti toward the organisation that had indentured his character told me he hated me for making him care. I had made his character feel for those he thought heartless and evil. It was this that made me think “Have I done it again?”. I must admit that the loss of Biondello sent a ripple of shock through us all. He was a solid sixth level summoner with a serpentine eidolon named Grestyl that had easily taken the characters down. But to have him poisoned as he fought to save those that he once had killed. It was a moving game.
In all honesty, I am not sure which had the most impact. It certainly feels like the most recent game, but it was only a few weeks back. I can still feel the sorrow at losing a favorite NPC and the elation of the feedback from the players. But will it have the same longevity as that game of Shadowrun in the 1990’s? Only time will tell…