The dorsal domain of the neural tube (prospective spinal cord and brain) is a highly dynamic region being first transiently populated by prospective neural crest (NC) cells that sequentially emigrate to generate most of the peripheral nervous system. Subsequently, it becomes the definitive roof plate (RP) of the central nervous system. The RP, in turn, is an inducer of dorsal interneuron development. The factors underlying establishment of the definitive RP and its segregation from NC and dorsal interneurons are currently unknown. We performed a transcriptome analysis comparing the dorsal neural tube at premigratory NC and RP stages. This unraveled a molecular heterogeneity between NC and RP stages. By implementing these genes, we asked whether Notch, an essential signaling pathway in development, is involved in RP formation. By gain and loss of Notch function in quail and mouse embryos, we provide evidence that Notch is required to establish the definitive RP, and to determine the choice between RP and interneuron fates, but not the segregation of RP from NC.