We found a novel lymphoid cell lineage, V alpha 14 NKT cell, which is characterized by 1) the expression of both NK1.1 (NK receptor) and an invariant TCR encoded by V alpha 14 and J alpha 281 gene segments; 2) the expression of unusual phenotypes, such as NK1.1+, B220+, Mac-1+, HSA+, CD44+, CD45Rlow and MEL-14low; and 3) the extrathymic development: V alpha 14 NKT cells appear at d9.5 of gestation before thymus development. Moreover, the deletion of the invariant V alpha 14 TCR gene expression caused the lack of NKT cells in vivo, while transgene of the invariant V alpha 14 V beta 8 TCR in the RAG-deficient background resulted in the generation of only V alpha 14 NKT cells without other lymphoid cells. These results indicate the essential requirement of invariant V alpha 14 TCR for the development of NKT cells. Recent studies clearly show that V alpha 14 NKT cells, but not NK cells or T cells are the primary target of IL-12 in the IL-12-mediated tumor rejection.