The adaptor protein SLP-76 is expressed in T lymphocytes and myeloid cells and is a substrate for ZAP-70 and Syk. We generated a SLP-76 null mutation in mice by homologous recombination in embryonic stem cells to evaluate the role of SLP-76 in T cell development and activation. SLP-76-deficient mice exhibited subcutaneous and intraperitoneal hemorrhaging and impaired viability. Analysis of lymphoid cells revealed a profound block in thymic development with absence of double-positive CD4⁺8⁺ thymocytes and of peripheral T cells. This block could not be overcome by in vivo treatment with anti-CD3. V-D-J rearrangement of the TCRβ locus was not obviously affected. B cell development was normal. These results indicate that SLP-76 collects all pre-TCR signals that drive the development and expansion of double-positive thymocytes.