Abstract The role of PI3K activity in T lymphocyte development is obscure because mice deficient in single PI3K catalytic subunits either die before birth (p110α−/− and p110β−/−) or lack a significant T cell developmental phenotype (p110γ−/− and p110δ−/−). We have generated mice deficient in both p110γ and p110δ and show that p110γ/δ−/− mice have a profound block in T cell development that occurs at the β-selection checkpoint. We show that pre-TCR-induced signaling is significantly reduced in p110γ/δ−/− thymocytes and that this results in a concomitant lack of proliferative expansion and increased apoptosis. The survival defect in p110γ/δ−/− thymocytes is associated with increased levels of the pro-apoptotic molecule Bcl2 interacting mediator of cell death. This work demonstrates that PI3K activity is critical for T cell development and depends on the combined function of p110γ and p110δ.