Effects of forced Hex expression on EB growth, Flk-1 expression, and BL-CFC development. (A-B) The impact of Hex forced expression on EB cell number. ES cells carrying a tet-inducible Hex or GFP cDNA were differentiated in serum in the presence or absence of Dox (1 μg/mL). Dox was added at day 0 (A) or day 2 (B) of the differentiation. Cells were counted at the indicated time points. The cultures were initiated with 1 × 10⁴ ES cells. The effect of forced Hex expression on the induction of Flk-1 (C) and BL-CFC development (D). tet-Hex ES cells were differentiated in the presence of serum with or without Dox (added time 0), and Flk-1 expression (C) and blast colonies formation (D) were examined at the indicated time points. Numbers for blast colony represent colonies per 2 × 10⁴ cells plated. (E) The effect of Hex on blast colony growth. Cells from uninduced EBs were cultured in the presence and absence of Dox (0.3 μg/mL) in the blast colony assay. Numbers for blast colonies are per 2 × 10⁴ cells plated. (F) tet-Hex ES cells were cultured with or without Dox (1 μg/mL) in serum-free conditions. Dox was added at day 2 of differentiation, and cells were counted at the indicated time points. The cultures were initiated with 1 × 10⁴ cells.