The development of CD1d-dependent natural killer T (NKT) cells is poorly understood. We have used both CD1d/alpha-galactosylceramide (CD1d/alphaGC) tetramers and anti-NK1.1 to investigate NKT cell development in vitro and in vivo. Confirming the thymus-dependence of these cells, we show that CD1d/alphaGC tetramer-binding NKT cells, including NK1.1(+) and NK1.1(-) subsets, develop in fetal thymus organ culture (FTOC) and are completely absent in nude mice. Ontogenically, CD1d/alphaGC tetramer-binding NKT cells first appear in the thymus, at day 5 after birth, as CD4(+)CD8(-)NK1.1(-)cells. NK1.1(+) NKT cells, including CD4(+) and CD4(-)CD8(-) subsets, appeared at days 7-8 but remained a minor subset until at least 3 wk of age. Using intrathymic transfer experiments, CD4(+)NK1.1(-) NKT cells gave rise to NK1.1(+) NKT cells (including CD4(+) and CD4(-) subsets), but not vice-versa. This maturation step was not required for NKT cells to migrate to other tissues, as NK1.1(-) NKT cells were detected in liver and spleen as early as day 8 after birth, and the majority of NKT cells among recent thymic emigrants (RTE) were NK1.1(-). Further elucidation of this NKT cell developmental pathway should prove to be invaluable for studying the mechanisms that regulate the development of these cells.