Progenitors of the first hematopoietic cells in the mouse arise in the early embryo from Brachyury-positive multipotent cells in the posterior-proximal region of the epiblast, but the mechanisms that specify primitive blood cells are still largely unknown. Pluripotency factors maintain uncommitted cells of the blastocyst and embryonic stem cells in the pluripotent state. However, little is known about the role played by these factors during later development, despite being expressed in the postimplantation epiblast. Using a dual transgene system for controlled expression at postimplantation stages, we found that Nanog blocks primitive hematopoiesis in the gastrulating embryo, resulting in a loss of red blood cells and downregulation of erythropoietic genes. Accordingly, Nanog-deficient embryonic stem cells are prone to erythropoietic differentiation. Moreover, Nanog expression in adults prevents the maturation of erythroid cells. By analysis of previous data for NANOG binding during stem cell differentiation and CRISPR/Cas9 genome editing, we found that Tal1 is a direct NANOG target. Our results show that Nanog regulates primitive hematopoiesis by directly repressing critical erythroid lineage specifiers.This work was supported by the Spanish government (grant BFU2014-54608-P and BFU2017-84914-P to MM; grants RYC-2011-09209 and BFU-2012-35892 to JI). The Gottgens and Nichols laboratories are supported by core funding from the Wellcome Trust and MRC to the Wellcome and MRC Cambridge Stem Cell Institute. The CNIC is supported by the Spanish Ministry of Science, Innovation and Universities (MINECO) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505)S
