Differentiation of hematopoietic stem and progenitors cells is an intricate process controlled in large part at the level of transcription. While some key megakaryocytic transcription factors have been identified, the complete network of megakaryocytic transcriptional control is poorly understood. Using global gene expression microarray analysis, Gene-Ontology-based functional annotations, and a novel inter-lineage comparison with parallel, isogenic granulocytic cultures as a negative control, we closely examined the mRNA level of transcriptional regulators in megakaryocytes derived from human mobilized peripheral blood CD34⁺ hematopoietic cells. This approach identified 199 differentially expressed transcription factors or transcriptional regulators. We identified and detailed the transcriptional kinetics of most known megakaryocytic transcription factors including GATA1, FLI1, and MAFG. Furthermore, many genes with transcription-factor activity or transcription-factor binding activity were identified in megakaryocytes that had not previously been associated with that lineage including BTEB1, NR4A2, FOXO1A, MEF2C, HDAC5, VDR, and several genes associated with the tumor suppressor p53 (HIPK2, FHL2, and TADA3L). Protein expression and nuclear localization was confirmed in megakaryocytic cells for three of the novel candidate megakaryocytic transcription factors: RFX5, MXD1, and FHL2. This work substantially expands our understanding of transcriptional regulation in megakaryocytic differentiation of stem and progenitor cells.