Hematopoietic differentiation of Klf6^-/- ES cells is impaired. (A) Cell-surface expression of Flk1 was analyzed by FACS. EBs differentiated for 2.5 to 5.5 days were stained with antibodies to Flk1, and the percentages of Flk1-positive cells are plotted. Reduced Flk1 expression is evident not only in Klf6^-/- ES cells (for up to 5 days), but also in Klf6^+/- cells. (B) Cells from day-4 EBs were subjected to blast colony-forming assay. EBs were trypsinized and 1 × 10⁵ cells were plated into methylcellulose cultures containing VEGF. Numbers of blast cell colonies were counted 4 days later. (C-E) Hematopoietic colony-forming assays from days 4, 5, and 6 EBs were performed. Cells from EBs were trypsinized and 1 × 10⁵ cells were plated into methylcellulose cultures containing a mix of cytokines (“Materials and methods”). Numbers of Ery^P (C), Mac (D), and Mix (E) colonies were counted. Bars represent SD of colony counts from 3 independent cultures. ^*P < .05 (Student t test).