The generation of specific cell types in a timely and orderly manner is a prerequisite for the formation of neural circuits during development. This process is largely governed by sequence-specific transcription factors, which bind to their cognate motifs on DNA to modulate gene expression. One such family of transcription factors known to regulate important developmental processes is the Nuclear factor one (NFI) family of transcription factors. Our analyses of cis-regulatory elements that were previously identified by the Mouse ENCODE project suggest that these transcription factors play a critical role in neuronal differentiation during cortical development. Specifically, we observed that the dimeric NFI motif, characterised by the palindromic sequence TTGGC(N)5GCCAA, is highly enriched at developmental enhancers derived from the embryonic forebrain. Our analyses demonstrate that the NFI motif is also the most abundant and most highly enriched transcription factor motif at enhancers during early neurogenesis – between E11.5 and E13.5 in the developing cortex. Ongoing analyses of the epigenome in Nfi knockout mouse models have demonstrated that these transcription factors contribute towards enhancer activation during cortical development. Gene ontology reveals a strong correlation between epigenomic changes and the developmental phenotypes observed in knockout mice and humans with NFIB haploinsufficiency. In summary, our work identifies a major regulator of enhancer activity during cortical development, and contributes towards identifying the downstream regulatory networks critical for neurogenesis in the developing brain.