Megakaryocytes are highly specialized precursor cells that differentiate to produce blood platelets via intermediate cytoplasmic extensions known as proplatelets. Recent advances in the understanding of megakaryocyte differentiation and platelet formation rely on a combination of genetic and cell biological studies with detailed structural analysis of cultured cells. Visualization of sequential steps in endomitosis has expanded our views on how megakaryocytes acquire polyploid DNA content, whereas studies in mouse models of platelet disorders provide clues into transcriptional pathways and those leading to the assembly of platelet-specific secretory granules. The experimental findings forge stronger links between cellular processes and molecular mechanisms, while observation of the underlying morphologic events in beginning to yield insights into the cytoskeletal mechanics of proplatelet formation. Here we review salient aspects of the emerging appreciation of the cellular and molecular basis of thrombopoiesis.