The final stages of of megakaryocyte (MK) maturation involve a series of steps, including polyploidization and proplatelet formation. Although these processes are highly dependent on dynamic changes in the microtubule (MT) cytoskeleton, the mechanisms responsible for regulation of MTs in MKs remain poorly defined. Stathmin is a highly conserved MT-regulatory protein that has been suggested to play a role in MK differentiation of human leukemic cell lines. However, previous studies defining this relationship have reached contradictory conclusions. In this study, we addressed this controversy and investigated the role of stathmin in primary human MKs. To explore the importance of stathmin down-regulation during megakaryocytopoiesis, we used a lentiviral-mediated gene delivery system to prevent physiologic down-regulation of stathmin in primary MKs. We demonstrated that sustained expression of constitutively active stathmin delayed cytoplasmic maturation (ie, glycoprotein GPIb and platelet factor 4 expression) and reduced the ability of MKs to achieve high levels of ploidy. Moreover, platelet production was impaired in MKs in which down-regulation of stathmin expression was prevented. These studies indicate that suppression of stathmin is biologically important for MK maturation and platelet production and support the importance of MT regulation during the final stages of thrombopoiesis.