Adenosine diphosphate (ADP) is a platelet agonist that causes platelet shape change and aggregation as well as generation of thromboxane A₂, another platelet agonist, through its effects on P2Y1, P2Y12, and P2X1 receptors. It is now reported that both 2-propylthio-D-βγ-dichloromethylene adenosine 5′-triphosphate (AR-C67085), a P2Y12 receptor–selective antagonist, and adenosine-2′-phosphate-5′-phosphate (A2P5P), a P2Y1 receptor–selective antagonist, inhibited ADP-induced thromboxane A₂ generation in a concentration-dependent manner, indicating that coactivation of the P2Y12 and P2Y1 receptors is essential for this event. SC49992, a fibrinogen receptor antagonist, blocked ADP-induced platelet aggregation and thromboxane A₂ production in a concentration-dependent manner. Similarly, P2 receptor antagonists or SC49992 blocked ADP-induced arachidonic acid liberation. Whereas SC49992 blocked arachidonic acid–induced platelet aggregation, it failed to inhibit thromboxane A₂ generation induced by arachidonic acid. Thus, ADP-induced arachidonic acid liberation, but not subsequent conversion to thromboxane A₂, requires outside-in signaling through the fibrinogen receptor. The Fab fragment of ligand-induced binding site–6 (LIBS6) antibody, which induces a fibrinogen-binding site on the integrin α_IIbβ₃, caused both platelet aggregation and thromboxane A₂ generation. Inhibitors of phosphoinositide 3-kinase, Syk, Src kinases, or protein tyrosine phosphatases inhibited platelet aggregation but not thromboxane A₂ generation, indicating that these signaling molecules have no significant role in phospholipase A₂ activation. In the presence of P2 receptor antagonists A2P5P or AR-C67085, LIBS6 failed to generate thromboxane A₂, suggesting that inside-out signaling through ADP receptors is necessary for this event. It was concluded that both outside-in signaling from the fibrinogen receptor and inside-out signaling from the P2Y1 and P2Y12 receptors are necessary for phospholipase A₂ activation, resulting in arachidonic acid liberation and thromboxane A₂ generation.