Hierarchy of transcription factor gene expression during ES cell differentiation. (A) Experimental strategy. Embryoid bodies were cultured for up to 3.75 days, and epiblast-like cells expressing neither marker (Bry⁻Flk1⁻), mesodermal cells expressing GFP only (Bry⁺Flk1⁻), and hemangioblast-enriched cell population expressing GFP and Flk-1 (Bry⁺Flk1⁺) were purified using fluorescence-activated cell sorting (FACS; supplemental Figure 1A). To obtain hematopoietic cells, we further cultured the (Bry⁺Flk1⁺) cells for 4 days under conditions supporting the generation of blast colonies and from these cultures isolated CD41⁺ cells, which have been shown to represent the first fully committed hematopoietic cells.³⁹  (B) Timing of mRNA expression of hematopoietic regulator genes (Runx1, Pu.1, and Csf1r) in differentiating ES cells. ES indicates ES cells; blast, 4-day blast cell culture derived from +/+ cells; LSK, Lin⁻Sca-1⁺c-kit^hi cells; CMP, common myeloid precursor cell; and Mac, primary macrophages. Numbers in panel B represent mean values of 2 independent experiments analyzed in duplicate.