Using genetic and phenotypic analyses, we have analyzed the developmental pathway of mouse CD1d-restricted invariant NKT cells. We provide strong evidence that similar to conventional T cells, positive selection of NKT cells occurs during a CD4(+)CD8(+) stage. Later stages of NKT cell development involved the down-regulation of both TCR and CD4 levels and therefore diverge from conventional T cell development pathways. A unique and complete dependency for development on Fyn, a Src family kinase member, also distinguishes the NKT cell and conventional T cell populations.