Platelets protect the vascular system during damage or inflammation, but platelet activation can result in pathological thrombosis. Activated platelets release a variety of extracellular vesicles (EVs). EVs shed from the plasma membrane often expose phosphatidylserine (PS). These EVs are pro-thrombotic and increased in number in many cardiovascular and metabolic diseases. The mechanisms by which PS-exposing EVs are shed from activated platelets are not well characterised. Cholesterol-rich lipid rafts provide a platform for coordinating signalling through receptors and Ca²⁺ channels in platelets. We show that cholesterol depletion with methyl-β-cyclodextrin or sequestration with filipin prevented the Ca²⁺-triggered release of PS-exposing EVs. Although calpain activity was required for release of PS-exposing, calpain-dependent cleavage of talin was not affected by cholesterol depletion. P2Y₁₂ and TPα, receptors for ADP and thromboxane A₂, respectively, have been reported to be in platelet lipid rafts. However, the P2Y₁₂ antagonist, AR-C69931MX, or the cyclooxygenase inhibitor, aspirin, had no effect on A23187-induced release of PS-exposing EVs. Together, these data show that lipid rafts are required for release of PS-exposing EVs from platelets.