The intracellular signaling pathways by which G protein–coupled receptors on the platelet surface initiate aggregation, a critical process for hemostasis and thrombosis, are not well understood. In particular, the contribution of the G_i pathway has not been directly addressed. We have investigated the activation of platelets from mice in which the gene for the predominant platelet Gα_i subtype, Gα_i2, has been disrupted. In intact platelets from Gα_i2-deficient mice, the inhibition of adenylyl cyclase by ADP was found to be partially impaired compared with wild-type platelets. Moreover, both ADP-dependent platelet aggregation and the activation of the integrin αIIbβ3 (GPIIb-IIIa) were strongly reduced in platelets from Gα_i2-deficient mice. In addition, Gα_i2-deficient platelets displayed impaired activation at low thrombin concentrations. This defect was mimicked by blocking the adenylyl cyclase–coupled platelet ADP receptor (P2Y₁₂) on wild-type platelets with a selective antagonist. These observations suggest that Gα_i2 is involved in the inhibition of platelet adenylyl cyclase in vivo and is a critical component of the signaling pathway for integrin activation by ADP, resulting in platelet aggregation. In addition, thrombin-dependent activation of mouse platelets is mediated, at least in part, by secreted ADP acting on the Gα_i2–linked ADP receptor.