A single hEB cell generates one blast colony with bipotential hematoendothelial capacity. (A) hEB cell dose response. To demonstrate that a single hEB cell has hemangioblastic capacity, the linear correlation of BL-CFC to the number of unfractionated Line H1 day 8 hEB plated was evaluated. Shown is the direct linear relationship observed with a slope ∼1, which is highly suggestive that each colony is derived from a single colony-forming cell. hEB cells from H1 hESC subclones transduced with lentiviral constructs expressing GFP or RFP demonstrated robust BL-CFC formation comparable with the H1 parental line (WT), with a similar linear correlation to numbers of cells plated. (B,C) GFP-RFP hEB Mixing Experiments. To further rule out the possibility that BL-CFC have apparent bipotentiality from the possibility that 2 separately derived progenitors are stuck or fused together, a 50:50 single-cell suspension mixture of GFP-expressing and RFP-expressing day 9 hEB cells was prepared (B), and blast colonies (phase image, “B” were expanded from this GFP-RFP mixture in BVF2H + TPO + IL-6, as before. (C) Secondary colonies from individually replated blast colonies (“B”) produced secondary endothelial colonies (“E”), hematopoietic colonies (“H”), or mixed hematoendothelial (“HE”) colonies. These secondary colonies were always GFP or RFP-expressing under fluorescent microscopy, and there were never colonies with both GFP and RFP expression mixed together. This experiment (with n = 24 picked blast colonies replated) was repeated with similar results, for a total of n = 50 colonies evaluated.