Ex vivo culture of hematopoietic stem and progenitor cells (HSPCs) under conditions designed to promote megakaryocytic (Mk) commitment, expansion, and differentiation has been proposed as means to produce mature megakaryocytes and platelets for transfusion. These cultures are highly sensitive to culture environment and supplements such as cytokines, though the intracellular mechanisms by which these factors affect the cells are not completely understood. In this study, we evaluate the global transcriptional profile of cells during primary human HSPC-initiated Mk culture and seek to identify novel genes and pathways involved in Mk differentiation. Three different culture conditions were tested: thrombopoietin (TPO) only, TPO with interleukin-3 and Flt3-ligand (cytokine cocktail), and TPO with nicotinamide. The time-course gene expression profiles were very consistent between culture conditions despite profound differences in expansion and maturation. Many known Mk and platelet-related genes were up-regulated as expected and several novel genes were identified which we suggest play previously unidentified roles in megakaryopoiesis. Interestingly, in contrast to the enhanced Mk maturation observed when nicotinamide is added to the cultures with TPO alone, the normal up-regulation of some known Mk and platelet genes, including P-selectin; RANTES; platelet glycoproteins Ib, V, VI, and IX; heparanase; and p21 was actually delayed or attenuated by NIC. Terminal Mk differentiation is intimately linked with a program of constitutive apoptosis. Decoupling this apoptosis from Mk maturation is critical for improving Mk culture efficiency but the molecular programs governing apoptosis in this cell-type are not understood. Gene ontology analysis led to the identification of several apoptosis-related genes that were consistently regulated under all three culture conditions. These included up-regulation of pro-apoptotic genes such as Bcl-2 binding component 3, tumor protein p53 inducible nuclear protein 1, and forkhead box O1A transcription factor and down-regulation of the anti-apoptotic BCL2. Using pathway analysis techniques, we found that these data imply that p53 signaling is modulated during differentiation. This result was confirmed during PMA-induced differentiation of a megakaryoblastic cell line (CHRF-288) using a p53-DNA binding ELISA. Together these data shed light on the gene expression patterns underlying the differentiation of HSPCs into megakaryocytes and provide leads for further study in the field of Mk cell culture engineering.