αPIX is a Rho GTPase guanine nucleotide exchange factor domain-containing signaling protein that associates with other proteins involved in cytoskeletal-membrane complexes. It has been shown that PIX proteins play roles in some immune cells, including neutrophils and T cells. In this study, we report the immune system phenotype of αPIX knockout mice. We extended αPIX expression experiments and found that whereas αPIX was specific to immune cells, its homolog βPIX was expressed in a wider range of cells. Mice lacking αPIX had reduced numbers of mature lymphocytes and defective immune responses. Antigen receptor-directed proliferation of αPIX⁻ T and B cells was also reduced, but basal migration was enhanced. Accompanying these defects, formation of T-cell-B-cell conjugates and recruitment of PAK and Lfa-1 integrin to the immune synapse were impaired in the absence of αPIX. Proximal antigen receptor signaling was largely unaffected, with the exception of reduced phosphorylation of PAK and expression of GIT2 in both T cells and B cells. These results reveal specific roles for αPIX in the immune system and suggest that redundancy with βPIX precludes a more severe immune phenotype.