FACS analysis of Venus knock-in ES cells differentiating on OP9 stromal cells. (A) Runx1^Venus/+ ES cells were cultured on OP9 cells and analyzed for the expression of Flk-1 and Venus by fluorescence-activated cell sorter (FACS) on the indicated days. (B) Flk-1⁺Venus⁺ cells were sorted on day 5.5 of differentiation and recultured on OP9 stroma. Expression of Flk-1, CD45, and TER119 on Venus⁺ cells was then analyzed using FACS on the indicated days. (C) Giemsa staining of Venus⁺ cells harvested on day 2.5 of differentiation of sorted Flk-1⁺Venus⁺ cells. All cells looked like immature hematopoietic cells of definitive hematopoietic origin. The image was captured with an Olympus DP50 microscope (× 40/0.75 NA objective lens) and DP control software (Olympus, Tokyo, Japan). (D) Flk-1⁺Venus^- cells were sorted on day 5.5 of differentiation and cultured again on OP9 stroma. On day 2.5 after sorting, cells were harvested and stained for Flk-1, CD45, and TER119 and were analyzed by flow cytometry. Results shown are representative of 3 independent experiments. Error bars indicate standard deviation of duplicate experiments.