Expression of hESC surface antigens and gene transcripts during stromal cell–mediated differentiation. (A) Flow cytometric analysis of hESCs differentiated on S17 stromal cells for 14 days demonstrates distinct populations of CD34^bright cells that costain with both CD31 and Flk1, and CD34^dim cells that coexpress CD45. Arrow shows histogram of CD34 expression (CD34-APC, shaded) of regions showing CD31⁺Flk1⁺ (top panel) and CD45⁺ (bottom panel) hESC-derived cells after 14 days of differentiation. Isotype control is shown in open histograms. Mean fluorescence intensity (MFI) of CD34 expression is indicated and demonstrates substantially higher CD34 expression in the CD34^brightCD31⁺Flk1⁺ cell population compared with the CD34^dimCD45⁺ cell population. (B) RT-PCR analysis of hematopoietic and endothelial gene expression during S17-mediated differentiation of hESCs over a time course, ranging from undifferentiated hESCs to day 21 of differentiation.