Sox7 at the onset of hematopoiesis specification. (A) Gene expression analysis by RT-PCR performed on day 3 EB subpopulations sorted for their respective expression of Bry-GFP and Flk1. (B) Expression analysis by RT-PCR on hemangioblast-derived blast colonies grown from sorted Flk1⁺ plated in semisolid clonogenic assay and harvested at the indicated time points. (C) Relative quantification of Sox7 expression by real-time PCR in the indicated population of Bry-GFP and Flk1 sorted at day 3 EBs and days 1 to 4 hemangioblast-derived blast colonies. Sorted Bry-GFP⁺Flk1⁻, mesodermal precursors were transduced with the indicated shRNA-expressing lentivirus, cultured for 2 days, and then tested for hematopoietic progenitors (D) and for endothelial potential (E) in clonogenic replating assay (n = 5). (F) Total cell recovery from mesodermal precursors transduced with the indicated virus after 2 days in culture. (G) Flow cytometric analysis of annexin V and 7-aminoactinomycin D staining on mesodermal precursors transduced by the indicated virus after 1 day of culture. All data are representative of at least 3 independent experiments. Error bars, where visible, represent SEM. *P < .01, **P < .05 compared with control shRNA.