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/DII-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(hl) NKT cells producing mainly interleukin-4 (IL-4), whereas NKT cells resembling CD44(hl) CD24(lo) liver NKT cells producing mainly interferon gamma (IFN-gamma) 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. (Blood. 2010; 115:230-237)