Embryonic stem cells (ESCs) cultured in leukemia inhibitory factor (LIF) plus 
fetal bovine serum (FBS) exhibit heterogeneity in the expression of naive and 
primed transcription factors. This heterogeneity reflects the dynamic condition 
of ESCs and their versatility to promptly respond to signaling effectors 
promoting naive or primed pluripotency. Here, we report that ESCs lacking Nanog 
or overexpressing Otx2 exhibit an early primed identity in LIF + FBS and fail to 
convert into 2i-induced naive state. Conversely, Otx2-null ESCs possess naive 
identity features in LIF + FBS similar to Nanog-overexpressing ESCs and convert 
poorly into FGF-induced early primed state. When both Nanog and Otx2 are 
inactivated, ESCs cultured in LIF + FBS exhibit primed identity and weakened 
ability to convert into naive state. These data suggest that, through mutual 
antagonism, NANOG and OTX2 specify the heterogeneous identity of ESCs cultured 
in LIF + FBS and individually predispose them for optimal response to naive or 
primed inducing factors.