Mutations in the MYH9 gene encoding nonmuscle myosin IIA lead to macrothrombocytopenia as observed in MYH9-related disorders. We used mice with megakaryocyte-restricted MYH9 inactivation to explore the role of myosin in thrombopoiesis. In situ, bone marrow MYH9Delta megakaryocytes were irregularly shaped, appearing leaky with poorly defined limits. The demarcation membranes were abnormally organized and poorly developed, pointing to an insufficient reservoir for the future formation of platelets. The cytoskeletal-rich peripheral zone was lacking due to the absence of the myosin filament network that normally surrounds the granular zone in wild-type cells. In vitro studies of cultured cells showed that MYH9Delta megakaryocytes were unable to form stress fibers upon adhesion to collagen, suggesting that the leaky shape results from defects in internal tension and anchorage to the extracellular environment. Surprisingly, the proportion of cells extending proplatelets was increased in MYH9Delta megakaryocytes and the proplatelet buds were larger. Overall, this study provides evidence for a role of myosin in different steps of megakaryocyte development through its participation in the maintenance of cell shape, formation and organization of the demarcation membranes and the peripheral zone, anchorage to the extracellular matrix, and proplatelet formation.