CD3 and CD3 are the most closely related CD3 components, both of which participate in the TCR–CD3 complex expressed on mature T cells. Interestingly, however, CD3 does not appear to participate functionally in the pre-T-cell receptor (TCR) complex that is expressed on immature T cells: disruption of CD3 gene expression has no effect on the developmental steps controlled by the pre-TCR. Here we report that in contrast with CD3, CD3 is an essential component of the pre-TCR. We generated mice selectively lacking expression of CD3, in which expression of CD3, CD3, CD3, pT and TCR remained undisturbed. Thus, all components for composing a pre-TCR are available, with the exception of CD3. Nevertheless, T-cell development is severely inhibited in CD3-deficient mice. The number of cells in the thymus is reduced to <1% of that in normal mice, and the large majority of thymocytes lack CD4 and CD8 and are arrested at the CD44-CD25+ double negative (DN) stage of development. Peripheral lymphoid organs are also practically devoid of T cells, with absolute numbers of peripheral T cells reduced to only 2–5% of those in normal mice. Both TCR and TCR lineages fail to develop effectively in CD3-deficient mice, although absence of CD3 has no effect on gene rearrangements of the TCR, and loci. Furthermore, absence of CD3 results in a severe reduction in the level of TCR and CD3 expression at the cell surface of thymocytes and peripheral T cells. The defect in the DN to double positive transition in mice lacking CD3 can be overcome by anti-CD3-mediated cross-linking. CD3 is thus essential for pre-TCR function.