Appropriate neural initiation of the pluripotent stem cells in the early embryos is critical for the development of the central nervous system. This process is regulated by the coordination of extrinsic signals and intrinsic programs. However, how the coordination is achieved to ensure proper neural fate commitment is largely unknown. Here, taking advantage of genome-wide ChIP-sequencing (ChIP-seq) and RNA-sequencing (RNA-seq) analyses, we demonstrate that the transcriptional factor Pou3f1 is an upstream activator of neural-promoting genes, and it is able to repress neural-inhibitory signals as well. Further studies revealed that Pou3f1 could directly bind neu-ral lineage genes like Sox2 and downstream targets of neural inhibition signaling such as BMP and Wnt. Our results thus identify Pou3f1 as a critical dual-regulator of the intrinsic transcription factors and the extrinsic cellular signals during neural fate commitment. Data were deposited in Gene Expression Omnibus (GEO) datasets under reference number GSE69865.