During neurogenesis, neural patterning is a critical step during which neural 
progenitor cells differentiate into neurons with distinct functions. However, 
the molecular determinants that regulate neural patterning remain poorly 
understood. Here we optimized the "dual SMAD inhibition" method to specifically 
promote differentiation of human pluripotent stem cells (hPSCs) into forebrain 
and hindbrain neural progenitor cells along the rostral-caudal axis. We report 
that neural patterning determination occurs at the very early stage in this 
differentiation. Undifferentiated hPSCs expressed basal levels of the 
transcription factor orthodenticle homeobox 2 (OTX2) that dominantly drove hPSCs 
into the "default" rostral fate at the beginning of differentiation. Inhibition 
of glycogen synthase kinase 3β (GSK3β) through CHIR99021 application sustained 
transient expression of the transcription factor NANOG at early differentiation 
stages through Wnt signaling. Wnt signaling and NANOG antagonized OTX2 and, in 
the later stages of differentiation, switched the default rostral cell fate to 
the caudal one. Our findings have uncovered a mutual antagonism between NANOG 
and OTX2 underlying cell fate decisions during neural patterning, critical for 
the regulation of early neural development in humans.