The cellular and molecular bases of platelet release by terminally differentiated megakaryocytes represent important questions in cell biology and hematopoiesis. Mice lacking the transcription factor NF-E2 show profound thrombocytopenia, and their megakaryocytes fail to produce proplatelets, the microtubule-based precursors of blood platelets. Using mRNA subtraction between normal and NF-E2–deficient megakaryocytes, cDNA was isolated encoding β1 tubulin, the most divergent β tubulin isoform. In NF-E2–deficient megakaryocytes, β1 tubulin mRNA and protein are virtually absent. The expression of β1 tubulin is exquisitely restricted to platelets and megakaryocytes, where it appears late in differentiation and localizes to microtubule shafts and coils within proplatelets. Restoring NF-E2 activity in a megakaryoblastic cell line or in NF-E2–deficient primary megakaryocytes rescues the expression of β1 tubulin. Re-expressing β1 tubulin in isolation does not, however, restore proplatelet formation in the defective megakaryocytes, indicating that other critical factors are required; indeed, other genes identified by mRNA subtraction also encode structural and regulatory components of the cytoskeleton. These findings provide critical mechanistic links between NF-E2, platelet formation, and selected microtubule proteins, and they also provide novel molecular insights into thrombopoiesis.