The development of CD1d-dependent natural killer T (NKT) cells is poorly understood. We have used both CD1d/α-galactosylceramide (CD1d/αGC) 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/αGC 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/αGC 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.