The signaling pathways that control T cell differentiation have only begun to be elucidated. Using T cell lines, it has been shown that class IA phosphatidylinositol 3-kinase (PI3K), a heterodimer composed of a p85 regulatory and a p110 catalytic subunit, is activated after TCR stimulation. Nonetheless, the contribution of p85/p110 PI3K isoforms in T cell development has not been described. Mice deficient in the other family of class I PI3K, p110gamma, which is regulated by G protein-coupled receptors, exhibit reduced thymus size. Here we examine T cell development in p110gamma-deficient mice and in mice expressing an activating mutation of the p85 regulatory subunit, p65(PI3K), in T cells. We show that p110gamma-deficient mice have a partial defect in pre-TCR-dependent differentiation, which is restored after expression of the p65(PI3K) activating mutation. Genetic alteration of both PI3K isoforms also affects positive selection; p110gamma deletion decreased and p65(PI3K) expression augmented the CD4(+)/CD8(+) differentiation ratio. Finally, data are presented showing that both PI3K isoforms influenced mature thymocyte migration to the periphery. These observations underscore the contribution of PI3K in T cell development, as well as its implication in determining the CD4(+)/CD8(+) T cell differentiation ratio in vivo.