Natural killer (NK) T cells are innate CD1d-restricted immune cells involved in regulation of immune tolerance, tumor immunity, and immunity to infectious pathogens. Human alpha-chain variable gene segment 24 (Valpha24) NK T cells exist in the periphery as two functionally distinct subsets: one CD4+ and one CD4- subset. However, the developmental pathway of human Valpha24 NK T cells is not well understood. Here, we show that Valpha24 NK T cells develop in the fetal thymus. The relative number of intrathymic NK T cell precursors decline in a linear manner with gestational age, and they are very rare in the neonatal thymus, indicating that these cells preferentially develop in the early fetal thymus. Their restriction element, CD1d, is expressed by a vast majority of thymocytes. A majority of intrathymic Valpha24 NK T cell progenitors are CD4+, whereas a minority are CD4/8(+/+). CD4+ Valpha24 NK T cell precursors show features of mature NK T cells, such as high levels of their semiinvariant T cell receptor and CD3 and some expression of CD161, whereas the CD4/8(+/+) precursors seem less mature. The cytokine IL-7 shows a biphasic effect on Valpha24 NK T cell progenitors in fetal thymic organ culture, with high doses driving proliferation of immature CD161-progenitors and low doses supporting survival and maturation. Thus, the data demonstrate that human Valpha24 NK T cells of the CD4+, but not the CD4-, subset develop in the early fetal thymus. Furthermore, data suggest an intrathymic pathway of CD4+ Valpha24 NK T cell development that is regulated by IL-7.