Hematopoietic cells develop from Scl-expressing cells. (A) VE-cadherin–expressing cells also express Flk-1 and Scl. Scl^+/hCD4 ES cells differentiated on OP9 for 6 days were subjected to FACS analyses for Flk-1, hCD4, and VE-cadherin expression. Based on the FACS data, the relationship between Flk-1–, Scl-, and VE-cadherin–expressing cells is presented in a Venn diagram (B). (C) VE-cadherin is not a marker for definitive hematopoietic cells. ES cells were differentiated on OP9 for 6 days, sorted for VE-cadherin^- and VE-cadherin⁺ cells, and replated. Subsequently, erythroid/macrophage mixed colonies were subjected to globin gene analyses as described.10 As shown, erythroid cells developing from both VE-cadherin^- and VE-cadherin⁺ cell populations expressed the β-major globin gene. N indicates H₂O, negative control; P, RNA from day 3 EBs, positive control. L32, ribosomal subunit protein is shown as a loading control.