Late depletion of Smad1 enhances erythroid potential correlating with enhanced expression levels for Gata1. (A) EBs induced on day 4 of differentiation examined for morphologic changes and GFP expression until day 6. EBs were transferred on day 7 to gelatinized tissue-culture plates and analyzed for their ability to adhere to the plate surface. (B) Hemangioblast marker gene expression was analyzed by qPCR; RNA from day 4–induced EBs was harvested after 24 hours, on day 5 of differentiation. Data were collected and analyzed using Gapdh as a reference control as in Figure 3B-E. Each experimental condition was repeated a minimum of 3 times and a representative result is shown. Error bars denote the SEM; **P < .01 compared with uninduced controls. (C) Flow cytometric analysis of FLK1/c-KIT expression at days 5 or 6 of EB differentiation, after induction of Smad1 depletion at day 4. (D) EryP colony-forming analysis of FLK1⁺ mesoderm sorted by flow cytometry from day 3.75 EBs to examine cell autonomy of Smad1 functions. Defined populations were recultured as EBs for a further 2 days with or without day 4 doxycycline treatment, and disaggregated to generate primitive erythroid colonies under the relevant permissive conditions. Each experimental condition was repeated 3 times, and a representative result is shown. Error bars denote SEM; *P < .05 compared with uninduced controls.