Cyclopamine treatment induces expression of definitive hematopoietic markers. (A-B) qPCR analysis of CD45⁺ cells sorted from control, cyclopamine-treated, and Shh-treated hEBs demonstrated a significant increase in HBB and lowered HBE on Hh inhibition. (C) hEBs grown in the presence or absence of cyclopamine at days 1-4 were plated into CFU assays and analyzed for HBB expression at day 15. HBB was augmented in bulk CFUs from cyclopamine-treated hEBs only. β-glucuronidase (GUSB) and GAPDH served as control genes. Bottom right: sequencing was used as a means to verify product specificity. Asterisks (*) present in a representative stretch of 16 bases indicate unique nucleotides present in HBB only; lack of any double sequence indicates amplicon purity. (D) Schematic representation of the protocol followed to derive erythroid cells from hEBs (“hemangioblast assay”). (E) Presence of anti-HBB immunoreactivity in control and cyclopamine treatment by flow cytometry. (F) Representative spectrum for HBB identified in cyclopamine-treated samples only. (G) Summary of globin hits relative to control β-actin in cyclopamine-treated versus control samples. Cord blood samples, run in parallel as a comparative control for developmental stage, are shown. A significant increase in HBG- and HBB-specific peptides was observed in cyclopamine-treated samples relative to paired hPSC-derived controls. **P < .01; ***P < .001.