In platelets, group IVA cytosolic phospholipase A₂ (cPLA₂α) has been implicated as a key regulator in the hydrolysis of platelet membrane phospholipids, leading to pro-thrombotic thromboxane A₂ and anti-thrombotic 12-(S)-hydroxyeicosatetranoic acid production. However, studies using cPLA₂α-deficient mice have indicated that other PLA₂(s) may also be involved in the hydrolysis of platelet glycerophospholipids. In this study, we found that group VIB Ca²⁺-independent PLA₂ (iPLA₂γ)-deficient platelets showed decreases in adenosine diphosphate (ADP)-dependent aggregation and ADP- or collagen-dependent thromboxane A₂ production. Electrospray ionization mass spectrometry analysis of platelet phospholipids revealed that fatty acyl compositions of ethanolamine plasmalogen and phosphatidylglycerol were altered in platelets from iPLA₂γ-null mice. Furthermore, mice lacking iPLA₂γ displayed prolonged bleeding times and were protected against pulmonary thromboembolism. These results suggest that iPLA₂γ is an additional, long-sought-after PLA₂ that hydrolyzes platelet membranes and facilitates platelet aggregation in response to ADP.