The SH2 domain containing leukocyte phosphoprotein of 76 kD (SLP-76) is critical for pre-TCR–mediated maturation to the CD4⁺CD8⁺ double positive (DP) stage in the thymus. The absolute block in SLP-76^null mice at the CD4⁻CD8⁻CD44⁻CD25⁺ (double-negative 3, DN3) stage has hindered our understanding of the role of this adaptor in αβ TCR-mediated signal transduction in primary thymocytes and peripheral T lymphocytes. To evaluate the requirements for SLP-76 in these events, we used a cre-loxP approach to generate mice that conditionally delete SLP-76 after the DN3 checkpoint. These mice develop DP thymocytes that express the αβ TCR on the surface, but lack SLP-76 at the genomic DNA and protein levels. The DP compartment has reduced cellularity in young mice and fails to undergo positive selection to CD4⁺ or CD8⁺ single positive (SP) cells in vivo or activation-induced cell death in vitro. A small number of CD4⁺SP thymocytes are generated, but these cells fail to flux calcium in response to an αβ TCR-generated signal. Peripheral T cells are reduced in number, lack SLP-76 protein, and have an abnormal surface phenotype. These studies show for the first time that SLP-76 is required for signal transduction through the mature αβ TCR in primary cells of the T lineage.