Adhesion to von Willebrand factor (VWF) induces platelet spreading, whereas adhesion to collagen induces aggregation. Here we report that cholesterol-rich domains (CRDs) or rafts play a critical role in clustering of receptors that control these responses. Platelets adhered to VWF and collagen show CRDs concentrated in filopodia which contain both the VWF receptor glycoprotein (GP) Ibalpha and the collagen receptor GPVI. Biochemical analysis of CRDs shows a threefold enrichment of GPIbalpha (but not GPVI) in VWF-adhered platelets and a fourfold enrichment of GPVI (but not GPIbalpha) in collagen-adhered platelets. Depletion of cholesterol (i) leaves the initial adhesion unchanged, (ii) inhibits spreading on VWF and aggregate formation on collagen, (iii) leaves filopodia formation intact, and (iv) reduces the localization in filopodia of GPIbalpha but not of GPVI. These data show that the adhesive substrate determines the composition of CRDs, and that cholesterol is crucial for redistribution of GPIbalpha but not of GPVI.