Establishment of a system with efficient generation of natural killer T (NKT) cells from embryonic stem (ES) cells would enable us to identify the cells with NKT-cell potential and obtain NKT cells with desired function. Here, using cloned ES (NKT-ES) cells generated by the transfer of nuclei from mature NKT cells, we have established a culture system that preferentially developed functional NKT cells and also identified early NKT progenitors, which first appeared on day 11 as a c-kit⁺ population in the cocultures on OP9 cells with expression of Notch ligand, delta-like1 (OP9/Dll-1) and became c-kit^lo/− on day 14. Interestingly, in the presence of Notch signals, NKT-ES cells differentiated only to thymic CD44^lo CD24^hi NKT cells producing mainly interleukin-4 (IL-4), whereas NKT cells resembling CD44^hi CD24^lo liver NKT cells producing mainly interferon γ (IFN-γ) and exhibiting strong adjuvant activity in vivo were developed in the switch culture starting at day 14 in the absence of Notch. The cloned ES culture system offers a new opportunity for the elucidation of the molecular events on NKT-cell development and for the establishment of NKT-cell therapy.