Mice that lack the matricellular angiogenesis inhibitor, thrombospondin-2 (TSP2), display a bleeding diathesis, despite normal blood coagulation and the lack of thrombocytopenia. Although platelets do not contain detectable levels of TSP2, TSP2-null platelets are compromised in their ability to aggregate in vivo in response to denudation of the carotid artery endothelium, and in vitro following exposure to adenosine diphosphate (ADP). Megakaryocytes (MKs) show high levels of TSP2 by immunohistochemical analysis of bone marrow. However, when cultured in vitro, MKs contain little TSP2 protein or mRNA. These findings suggest that most TSP2 is acquired from the bone marrow microenvironment. Consistent with this hypothesis, MKs take up recombinant TSP2 in an integrin-dependent manner when it is supplied in the culture medium. Furthermore, uptake of TSP2 in vitro affects MK differentiation and proplatelet formation. The functional significance of this process is supported by the presence of ultrastructural abnormalities in TSP2-null bone marrow, including extensive fragmentation of the peripheral zone in MKs and failure of this zone to form close associations with vascular sinuses. We conclude that the uptake of TSP2 by MKs from the marrow milieu is required for proper MK function and the release of functionally competent platelets.