ESC-derived VE-cadherin⁺CD45⁻ cells generate primitive and definitive erythrocytes sequentially. Immunostaining of floating erythrocytes on days 10 + 6 (A-E) and 10 + 30 (F-J). Cy3 detection of erythrocytes stained with anti–α-, anti–β-, anti–ϵ-, anti–γ-, or anti–ζ-globin mAbs (red) and FITC detection with anti–Hb polyclonal Ab (green). Anti-Hb Ab, which reacts with embryonic, fetal, and adult erythrocytes, was used to detect all erythrocytes. Nuclei were labeled with Hoechst 33342. Merged images are shown. Original magnification × 200. Scale bars, 50 μm. (K-L) Sequential analysis of the proportion of erythrocytes positive for anti–α- or anti–ζ-globin mAbs (K) and anti–β-, anti–γ-, or anti–ϵ-globin mAbs (L) among the total erythrocytes. Data are presented as mean ± SD of 3 independent experiments.