Breast cancer is the most common tumor among women with inherited mutations in the p53 gene (Li-Fraumeni syndrome). The tumors represent the basal-like subtype, which has been suggested to originate from mammary stem/progenitor cells. In mouse mammary epithelium, mammosphere-forming potential was increased with decreased dosage of the gene encoding the p53 tumor suppressor protein (Trp53). Limiting dilution transplantation also showed a 3.3-fold increase in the frequency of long-term regenerative mammary stem cells in Trp53−/− mice. The repression of mammospheres by p53 was apparent despite the absence of apoptotic responses to radiation indicating a dissociation of these two activities of p53. The effects of p53 on progenitor cells were also observed in TM40A cells using both mammosphere-forming assays and the DsRed-let7c-sensor. The frequency of long-term label-retaining epithelial cells was decreased in Trp53−/− mammary glands indicating that asymmetric segregation of DNA is diminished and contributes to the expansion of the mammary stem cells. Treatment with an inhibitor of γ-secretase (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester) reduced the number of Trp53−/− mammospheres to the level found in Trp53+/+ cells. These results demonstrate that basal levels of p53 restrict mammary stem/progenitor cells through Notch and that the Notch pathway is a therapeutic target to prevent expansion of this vulnerable pool of cells. STEM CELLS 2011;29:119–127