Megakaryocytes, among the rarest of hematopoietic cells, serve the essential function of producing numerous platelets. Genetic studies have recently provided rich insights into the molecular and transcriptional regulation of megakaryocyte differentiation and thrombopoiesis. Three transcription factors, GATA-1, FOG-1, and NF-E2, are essential regulators of distinct stages in megakaryocyte differentiation, extending from the birth of early committed progenitors to the final step of platelet release; a fourth factor, Fli-1, likely also plays an important role. The putative transcriptional targets of these regulators, including the NF-E2-dependent hematopoietic-specific β-tubulin isoform β1, deepen our understanding of molecular mechanisms in platelet biogenesis. The study of rare syndromes of inherited thrombocytopenia in mice and man has also refined the emerging picture of megakaryocyte maturation. Synthesis of platelet-specific organelles is mediated by a variety of regulators of intracellular vesicle membrane fusion, and platelet release is coordinated through extensive and dynamic reorganization of the actin and microtubule cytoskeletons. As in other aspects of hematopoiesis, characterization of recurrent chromosomal translocations in human leukemias provides an added dimension to the molecular underpinnings of megakaryocyte differentiation. Long regarded as a mysterious cell, the megakaryocyte is thus yielding many of its secrets, and mechanisms of thrombopoiesis are becoming clearer. Although this review focuses on transcriptional control mechanisms, it also discusses recent advances in broader consideration of the birth of platelets.