CD3γ and CD3δ are two highly related components of the T cell receptor (TCR)-CD3 complex which is essential for the assembly and signal transduction of the T cell receptor on mature T cells. In gene knockout mice deficient in either CD3δ or CD3γ, early thymic development mediated by pre-TCR was either undisturbed or severely blocked, respectively, and small numbers of TCR-αβ+ T cells were detected in the periphery of both mice. γδ T cell development was either normal in CD3δ−/− mice or partially blocked in CD3γ−/− mice. To examine the collective role of CD3γ and CD3δ in the assembly and function of pre-TCR and in the development of γδ T cells, we generated a mouse strain with a disruption in both CD3γ and CD3δ genes (CD3γδ−/−). In contrast to mice deficient in either CD3γ or CD3δ chains, early thymic development mediated by pre-TCR is completely blocked, and TCR-αβ+ or TCR-γδ+ T cells were absent in the CD3γδ−/− mice. Taken together, these studies demonstrated that CD3γ and CD3δ play an essential, yet partially overlapping, role in the development of both αβ and γδ T cell lineages.