Application of dendritic cells (DCs) to prime responses to tumor Ags provides a promising approach to immunotherapy. However, only a limited number of DCs can be manufactured from adult precursors. In contrast, pluripotent embryonic stem (ES) cells represent an inexhaustible source for DC production, although it remains a major challenge to steer directional differentiation because ES cell-derived cells are typically immature with impaired functional capacity. Consistent with this notion, we found that mouse ES cell-derived DCs (ES-DCs) represented less mature cells compared with bone marrow-derived DCs. This finding prompted us to compare the gene expression profile of the ES cell- and adult progenitor-derived, GM-CSF-instructed, nonconventional DC subsets. We quantified the mRNA level of 17 DC-specific transcription factors and observed that 3 transcriptional regulators (Irf4, Spi-B, and Runx3) showed lower expression in ES-DCs than in bone marrow-derived DCs. In light of this altered gene expression, we probed the effects of these transcription factors in developing mouse ES-DCs with an isogenic expression screen. Our analysis revealed that forced expression of Irf4 repressed ES-DC development, whereas, in contrast, Runx3 improved the ES-DC maturation capacity. Moreover, LPS-treated and Runx3-activated ES-DCs exhibited enhanced T cell activation and migratory potential. In summary, we found that ex vivo-generated ES-DCs had a compromised maturation ability and immunogenicity. However, ectopic expression of Runx3 enhances cytokine-driven ES-DC development and acts as an instructive tool for the generation of mature DCs with enhanced immunogenicity from pluripotent stem cells.