Serum response factor (SRF) is a ubiquitously expressed transcription factor that binds DNA at CArG (CC[A/T]₆GG) domains in association with myocardin-family proteins (eg, myocardin-related transcription factor A [MRTFA]) or the ternary complex factor family of E26 transformation-specific (ETS) proteins. In primary hematopoietic cells, knockout of either SRF or MRTFA decreases megakaryocyte (Mk) maturation causing thrombocytopenia. The human erythroleukemia (HEL) cell line mimics the effects of MRTFA on Mk maturation, and MRTFA overexpression (MRTFA^OE) in HEL cells enhances megakaryopoiesis. To identify the mechanisms underlying these effects, we performed integrated analyses of anti-SRF chromatin immunoprecipitation (ChIP) and RNA-sequencing data from noninduced and phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA])–induced HEL cells, with and without MRTFA^OE. We found that 11% of genes were upregulated with TPA induction, which was enhanced by MRTFA^OE, resulting in an upregulation of 25% of genes. MRTFA^OE increased binding of SRF to genomic sites and enhanced TPA-induced expression of SRF target genes. The TPA-induced genes are predicted to be regulated by SRF and ETS factors, whereas those upregulated by TPA plus MRTFA^OE lack ETS binding motifs, and MRTFA^OE skews SRF binding to genomic regions with CArG sites in regions relatively lacking in ETS binding motifs. Finally, ChIP–polymerase chain reaction using HEL cells and primary human CD34⁺ cell–derived subpopulations confirms that both SRF and MRTFA have increased binding during megakaryopoiesis at upregulated target genes (eg, CORO1A). We show for the first time that MRTFA increases both the genomic association and activity of SRF and upregulates genes that enhance primary human megakaryopoiesis.