Abstract CTLA-4 and PD-1 are receptors that negatively regulate T cell activation. Ligation of both CTLA-4 and PD-1 blocked CD3/CD28 mediated upregulation of glucose metabolism and Akt activity, but each accomplished this regulation using separate mechanisms. CTLA-4 mediated inhibition of Akt phosphorylation is sensitive to okadaic acid, providing direct evidence that PP2A plays a prominent role in mediating CTLA-4 suppression of T cell activation. In contrast, PD-1 signaling inhibits Akt phosphorylation by preventing CD28 mediated activation of phosphatidylinositol 3-kinase (PI3K). The ability of PD-1 to suppress PI3K/Atk activation was dependent upon the ITSM located in its cytoplasmic tail. Lastly, PD-1 ligation is more effective in suppressing CD3/28 induced changes in the T cell transcriptional profile, suggesting that differential regulation of PI3K activation by PD-1 and CTLA-4 ligation results in distinct cellular phenotypes. Together, these data suggest that CTLA-4 and PD-1 inhibit T cell activation through distinct and potentially synergistic mechanisms.