In this study, to investigate whether presenilin 1 (PS1) plays essential physiological roles in human cortical mature neurons, we generated PS1 conditional knockout (cKO) induced pluripotent stem cells (iPSCs), in which PS1 can be ablated selectively under an introduction of Cre recombinase, and/or additional PS2 KO iPSC. We then differentiated the fPS1/fPS1 and fPS1/fPS1;PS2-/- iPSCs into human cortical neurons in vitro, and ablated PS1 proteins by infection of lentivirus expressing Cre. Whereas no gross morphological alteration and neuronal marker expression was observed between PS1- and/or PS2-null neurons and control neurons, Aβ production was robustly reduced only in PS1/PS2-null neurons. In contrast to previous studies using mouse genetics, in which γ-secretase activity is mainly attributable to PS1, human PS2/γ-secretase activity is unexpectedly comparable with PS1/γ-secretase in human neurons. These results suggest an importance of enzyme/substrate subcellular localization.