The prion protein is infamous for its involvement in a group of neurodegenerative diseases known as Transmissible Spongiform Encephalopathies. In the longstanding quest to decipher the physiological function of its cellular isoform, PrP C, the discovery of its participation to the selfrenewal of hematopoietic and neural stem cells has cast a new spotlight on its potential role in stem cell biology. However, still little is known on the cellular and molecular mechanisms at play. Here, by combining in vitro and in vivo murine models of PrP C depletion, we establish that PrP C deficiency severely affects the Notch pathway, which plays a major role in neural stem cell maintenance. We document that the absence of PrP C in a neuroepithelial cell line or in primary neurospheres is associated with drastically reduced expression of Notch ligands and receptors, resulting in decreased levels of Notch target genes. Similar alterations of the Notch pathway are recovered in the neuroepithelium of Prnp(-/-) embryos during a developmental window encompassing neural tube closure. In addition, in line with Notch defects, our data show that the absence of PrP C results in altered expression of Nestin and Olig2 as well as Ncadherin distribution. We further provide evidence that PrP C controls the expression of the epidermal growth factor receptor (EGFR) downstream from Notch. Finally, we unveil a negative feedback action of EGFR on both Notch and PrP C. As a whole, our study delineates a molecular scenario through which PrP C takes part to the self-renewal of neural stem and progenitor cells.