Platelets are an abundant source of CD40 ligand (CD154), an immunomodulatory and proinflammatory molecule implicated in the onset and progression of several inflammatory diseases, including systemic lupus erythematosus (SLE), diabetes, and cardiovascular disease. Heretofore considered largely restricted to activated T cells, we initiated studies to investigate the source and regulation of platelet-associated CD154. We found that CD154 is abundantly expressed in platelet precursor cells, megakaryocytes. We show that CD154 is expressed in primary human CD34+ and murine hematopoietic precursor cells only after cytokine-driven megakaryocyte differentiation. Furthermore, using several established megakaryocyte-like cells lines, we performed promoter analysis of the CD154 gene and found that NFAT, a calcium-dependent transcriptional regulator associated with activated T cells, mediated both differentiation-dependent and inducible megakaryocyte-specific CD154 expression. Overall, these data represent the first investigation of the regulation of a novel source of CD154 and suggests that platelet-associated CD154 can be biochemically modulated.