Mouse embryonic stem cells (ESCs) and the inner cell mass (ICM)-derived epiblast 
exhibit naive pluripotency. ESC-derived epiblast stem cells (EpiSCs) and the 
postimplantation epiblast exhibit primed pluripotency. Although core 
pluripotency factors are well-characterized, additional regulators, including 
Otx2, recently have been shown to function during the transition from naive to 
primed pluripotency. Here we uncover a role for Otx2 in the control of the naive 
pluripotent state. We analyzed Otx2-binding activity in ESCs and EpiSCs and 
identified Nanog, Oct4, and Sox2 as direct targets. To unravel the Otx2 
transcriptional network, we targeted the strongest Otx2-binding site in the 
Nanog promoter, finding that this site modulates the size of specific 
ESC-subtype compartments in cultured cells and promotes Nanog expression 
in vivo, predisposing ICM differentiation to epiblast. Otx2-mediated Nanog 
regulation thus contributes to the integrity of the ESC state and cell lineage 
specification in preimplantation development.