Abstract An effect of thyroid hormone on erythropoiesis has been known for more than a century but the molecular mechanism(s) by which thyroid hormone affects red cell formation are still elusive. Our study demonstrates an essential role of thyroid hormone during terminal human erythroid cell differentiation; specific depletion of thyroid hormone from the culture medium completely blocked human erythroid differentiation. Genome wide analysis showed that thyroid hormone receptor β (TRβ) occupies many gene loci encoding transcripts abundant during terminal erythropoiesis, including globin genes, and cooperates with GATA-1 and RNA polymerase II (Pol II) to regulate their expression. Treatment with TRβ agonists enhanced erythroblast differentiation in vivo and alleviated anemic symptoms in a chronic anemia mouse model. To identify factors that cooperate with TRβ during human erythroid terminal differentiation, we conducted RNA-Seq in human reticulocytes and identified NCOA4 as a critical regulator of terminal differentiation. Furthermore, Ncoa4-/- mice are anemic in both the embryonic and perinatal periods and fail to respond to thyroid hormone by enhanced erythropoiesis. Genome wide analysis suggests that thyroid hormone promotes NCOA4 recruitment to chromatin regions that are in proximity to Pol II and are highly associated with transcripts abundant during terminal differentiation. Collectively, our results reveal the molecular mechanism of thyroid hormone function on red blood cell formation and are potentially useful to treat certain anemias. Disclosures No relevant conflicts of interest to declare.