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MicroRNAs (miRNAs) can control stem cell differentiation by targeting mRNAs. Using 96‐well plate electroporation, we screened 466 human miRNA mimics by four‐color flow cytometry to explore differentiation of common myeloid progenitors (CMP) derived from human embryonic stem cells (hESCs). The transfected cells were then cultured in a cytokine cocktail that supported multiple hematopoietic lineages. At 4–5 days post‐transfection, flow cytometry of erythroid (CD235 + CD41 − ), megakaryocyte (CD41 + CD42 + ), and myeloid (CD18 + CD235 − ) lineages revealed miR‐105 as a novel enhancer of megakaryocyte production during in vitro primitive hematopoiesis. In hESC‐derived CMPs, miR‐105 caused a sixfold enhancement in megakaryocyte production. miR‐513a, miR‐571, and miR‐195 were found to be less potent megakaryocyte enhancers. We confirmed the relevance of miR‐105 in adult megakaryopoiesis by demonstrating increased megakaryocyte yield and megakaryocyte colony forming potential in human adult CD34 +  cells derived from peripheral blood. In addition, adult CD34 +  cells express endogenous miR‐105 during megakaryocyte differentiation. siRNA knockdown of the hematopoietic transcription factor c‐Myb caused a similar enhancement of megakaryocyte production as miR‐105. Finally, a luciferase/c‐Myb‐3′UTR construct and Western blot analysis demonstrated that the hematopoietic transcription factor c‐Myb mRNA was a target of miR‐105. We report a novel hESC‐based miR screening platform and demonstrate that miR‐105 is an enhancer of megakaryopoiesis in both primitive and definitive hematopoiesis. S tem  C ells   2014;32:1337–1346

MicroRNA Screen of Human Embryonic Stem Cell Differentiation Reveals miR‐105 as an Enhancer of Megakaryopoiesis from Adult CD34+ Cells