Human embryonic stem cell (hESC)-derived hematopoietic stem/progenitor cells hold tremendous potential as alternative cell sources for the treatment of various hematological diseases, drug discovery and toxicological screening. However, limited number of hematopoietic stem/progenitor cells generated from the differentiation of hESCs hinders their downstream applications. Here, we show that aryl hydrocarbon receptor antagonist StemRegenin 1 (SR1) selectively promotes expansion of hESC-derived lin CD34 hematopoietic progenitors in a concentration-dependent manner. The colony-forming cell (CFC) activity was found to be enriched in the CD34 cells that were expanded with SR1; however, these cells have less colony-forming activity as compared to unexpanded cells (1,338 vs. 7 of CD34 cells to form 1 colony, respectively). Interestingly, SR1 showed a bipotential effect on the proliferation of CD34 negative population, that is low dose of SR1 (1 µM) enhanced cell proliferation, whereas it was repressed at higher doses (>5 µM). In summary, our results suggest that SR1 has the potential to facilitate expansion of hESC-derived lin CD34 hematopoietic progenitors, which further retain the potential to form multilineage hematopoietic colonies.