Mammary epithelium comprises a layer of luminal cells and a basal myoepithelial cell layer. Both mammary epithelial compartments, basal and luminal, contain stem and progenitor cells, but only basal cells are capable of gland regeneration upon transplantation. Aberrant expansion of stem/progenitor cell populations is considered to contribute to breast tumorigenesis. Germline deletions of p53 in humans and mice confer a predisposition to tumors, and stem cell frequency is abnormally high in the mammary epithelium of p53-deficient mice. However, it is unknown whether stem/progenitor cell amplification occurs in both, basal and luminal cell populations in p53-deficient mammary tissue. We used a conditional gene deletion approach to study the role of p53 in stem/progenitor cells residing in the mammary luminal and basal layers. Using two- and three-dimensional cell culture assays, we showed that p53 loss led to the expansion of clonogenic stem/progenitor cells in both mammary epithelial cell layers. Moreover, following p53 deletion, luminal and basal stem/progenitor cells acquired a capacity for unlimited propagation in mammosphere culture. Furthermore, limiting dilution and serial transplantation assays revealed amplification and enhanced self-renewal in the basal regenerating cell population of p53-deficient mammary epithelium. Our data suggest that the increase in stem/progenitor cell activity may be, at least, partially mediated by the Notch pathway. Taken together, these results strongly indicate that p53 restricts the propagation and self-renewal of stem/progenitor cells in both layers of the mammary epithelium providing further insight into the impact of p53 loss in breast cancerogenesis.