To determine which CD3 components are required for early T cell development, we generated mice with a targeted mutation of the CD3-epsilon gene and characterized their T cell populations relative to those found in CD3-zeta/eta-and recombinase activating gene (RAG)-deficient mice. In the absence of intact CD3-epsilon subunit, thymocytes do not progress beyond the CD44-/lowCD25+ triple-negative stage and appear to be arrested at the very same developmental control point as RAG-deficient thymocytes. In contrast, the disruption of the CD3-epsilon/eta gene does not totally abrogate the progression through this control point. CD3-epsilon-deficient thymocytes do rearrange their T cell receptor (TCR) beta gene segments and produce low levels of full-length TCR beta transcripts. Taken together, these results establish an essential role for the CD3-epsilon gene products during T cell development and further suggest that the CD3-epsilon polypeptides start to exert their function as part of a pre-TCR through which CD44-/lowCD25+ triple-negative cells monitor the occurrence of productive TCR beta gene rearrangements. Finally, the absence of intact CD3-epsilon polypeptides had no discernible effect on the completion of TCR gamma and TCR delta gene rearrangements, emphasizing that they are probably not subjected to the same epigenetic controls as those operating on the expression of TCR alpha and beta genes.