Platelets are essential in maintaining hemostasis following inflammation or injury to the vasculature. Dysregulated platelet activity often results in thrombotic complications leading to myocardial infarction and stroke. Activation of the FcγRIIa receptor leads to immune-mediated thrombosis, which is often life threatening in patients undergoing heparin-induced thrombocytopenia or sepsis. Inhibiting FcγRIIa-mediated activation in platelets has been shown to limit thrombosis and is the principal target for prevention of immune-mediated platelet activation. In this study, we show for the first time that platelet 12(S)-lipoxygenase (12-LOX), a highly expressed oxylipin-producing enzyme in the human platelet, is an essential component of FcγRIIa-mediated thrombosis. Pharmacologic inhibition of 12-LOX in human platelets resulted in significant attenuation of FcγRIIa-mediated aggregation. Platelet 12-LOX was shown to be essential for FcγRIIa-induced phospholipase Cγ2 activity leading to activation of calcium mobilization, Rap1 and protein kinase C activation, and subsequent activation of the integrin αIIbβ3. Additionally, platelets from transgenic mice expressing human FcγRIIa but deficient in platelet 12-LOX, failed to form normal platelet aggregates and exhibited deficiencies in Rap1 and αIIbβ3 activation. These results support an essential role for 12-LOX in regulating FcγRIIa-mediated platelet function and identifies 12-LOX as a potential therapeutic target to limit immune-mediated thrombosis.