Significance Invariant natural killer T (iNKT) cells participate in the first line of body defense and can be classified into three functional subsets. Differences in their developmental and functional control further contribute to their functional complexity. Here, we show that Foxo1 promotes survival of iNKT1 and iNKT2 cells at early stages of differentiation but is dispensable for iNKT17 cell development due to its distinct contributions to IL7R expression in these subsets. Additionally, Foxo1 is required for mTORC1 activation and function of iNKT1 and iNKT2 cells through inhibiting TSC1–TSC2 interaction but not for iNKT17 cells. Our study proposes that Foxo1 distinctly controls development and functions of different iNKT subsets, and targeting Foxo1 would lead to a functional shift of iNKT cells.