The molecular mechanisms governing gamma-globin expression in a subset of fetal hemoglobin (alpha₂:gamma₂; HbF) expressing red blood cells (F-cells) and the mechanisms underlying the variability of response to hydroxyurea induced gamma-globin expression in the treatment of sickle cell disease are not completely understood. To explore molecular differences in these conditions, a serum-free in vitro culture system suitable for large scale production of erythroblasts derived from primary human hematopoietic progenitors is optimized. The culture system recapitulates steady-state adult erythropoiesis and can support erythroid differentiation with the addition of cytotoxic hydroxyurea. Using this system, intra-person clonal populations of erythroblasts derived from bone marrow common myeloid progenitors were evaluated for molecular factors associated with gamma-globin expression. Data demonstrate that the level of fetal hemoglobin produced in F-cells negatively correlates with expression of BCL11A, KLF1 and TALL With the addition of hydroxyurea, successful induction of gamma-globin includes a further reduction in BCL11 A, KLF1 and TALI expression along with a decrease in SOX6 expression. These data suggests that expression changes in this transcription factor network modulate gamma-globin expression in F-cells during steady state erythropoiesis and after induction with hydroxyurea.