Dynamin 3 (DNM3) is a member of a family of motor proteins that participate in a number of membrane rearrangements such as cytokinesis, budding of transport vesicles, phagocytosis, and cell motility. Recently, DNM3 was implicated as having a role in megakaryocyte (MK) development. To further investigate the functional role of DNM3 during megakaryocytopoiesis, we introduced sequence-specific short hairpin RNAs (shRNAs) into developing MKs. The results showed that knockdown of DNM3 inhibited a stage of MK development that involved progenitor amplification. This was evident by significant decreases in the number of colony forming unit-megakaryocytes, the total number of nucleated cells, and the number of CD41(+) and CD61(+) MKs produced in culture. Using a styrl membrane dye to quantify the demarcation membrane system (DMS) of terminally differentiated MKs, we found that DNM3 co-localized with the DMS and that DNM3 lentiviral shRNAs precluded the formation of the DMS. Knockdown of dynamin 3 in murine MKs also caused a decrease in the number of morphologically large MKs and the overall size of large MKs was decreased relative to controls. MK protein lysates were used in overlay blots to show that both DNM3 and actin bind to nonmuscle myosin IIA (MYH9). Consistent with these observations, immunofluorescence studies of MKs and proplatelet processes showed co-localization of DNM3 with MYH9. Overall, these studies demonstrate that DNM3 not only participates in MK progenitor amplification, but is also involved in cytoplasmic enlargement and the formation of the DMS.