The invariant NKT (iNKT) cell lineage contains CD4+ and CD4− subsets. The mechanisms that control such subset differentiation and iNKT cell maturation in general have not been fully understood. RasGRP1, a guanine nucleotide exchange factor for TCR-induced activation of the Ras–ERK1/2 pathway, is critical for conventional αβ T cell development but dispensable for generating regulatory T cells. Its role in iNKT cells has been unknown. In this study, we report severe decreases of iNKT cells in RasGRP1−/− mice through cell intrinsic mechanisms. In the remaining iNKT cells in RasGRP1−/− mice, there is a selective absence of the CD4+ subset. Furthermore, RasGRP1−/− iNKT cells are defective in TCR-induced proliferation in vitro. These observations establish that RasGRP1 is not only important for early iNKT cell development but also for the generation/maintenance of the CD4+ iNKT cells. Our data provide genetic evidence that the CD4+ and CD4− iNKT cells are distinct sublineages with differential signaling requirements for their development.