Highly conserved transcription factors play important roles in the development of specific tissues and organs across multiple species. Pax6 is one such factor: its expression is both highly conserved and absolutely required in the development of the vertebrate central nervous system. Despite its conservation, however, the role of Pax6 in regulating neural progenitor activity in the developing brain differs between mouse and human, although the evolutionary origin and mechanism of this functional flexibility remain unclear. Now, Tadashi Nomura and colleagues identify conserved and divergent functions for Pax6 in the developing chick and mouse pallium. The authors perform in vivo targeting of Pax6 by CRISPR/Cas9-mediated genome editing and show that it plays invariable roles in brain patterning and progenitor proliferation in the developing chick dorsal pallium, a region homologous to the mammalian neocortex. By contrast, the authors demonstrate that stage-specific neurogenic functions of Pax6 are unique to mammals, in agreement with temporal changes in the regulation of Notch signalling. Overexpression of Pax6 in the developing chick pallium identifies Dbx1 as a likely target, and the authors further show that the cis-regulatory region of Dbx1 acts as a conserved cryptic enhancer in response to Pax6, although Dbx1 expression is highly diversified in amniote brains.