Thrombospondin-1 (TSP-1) is an inhibitor of angiogenesis, inducing apoptosis of the endothelial cells via CD36 signaling mechanism. We investigated CD36 expression and the effect of TSP-1 on megakaryocytopoiesis, with and without pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), and with and without blocking TSP-1 binding with receptor CD36 on megakaryocytic cells. Our data showed that TSP-1 induced a dose-dependent growth inhibition in both murine and human colony forming unit-megakaryocyte (CFU-MK) assays and significantly counteracted the mitogenic effect from PEG-rHuMGDF. Moreover, the growth suppression induced by TSP-1 was correlated with CD36 expression in megakaryocytic cell lines, where growth inhibition was demonstrated in CD36 positive (Meg-01, Dami and CHRF-288-11) but not in CD36 negative (M-07e) cell lines. More importantly, the inhibitory effect of TSP-1 on both human CFU-MK and Meg-01 cells was partially but significantly reversed by the addition of FA6-152 (anti-CD36), a blocking antibody which blocks the access of TSP-1 to CD36 receptor, suggesting that the TSP-1-induced inhibition of megakaryocytopoiesis is probably mediated in part by the binding of TSP-1 to CD36 expressed on the megakaryocytic progenitors. Thus, our findings represent the first demonstration that TSP-1 inhibits in vitro megakaryocytopoiesis via interaction with CD36.