Using a syngeneic p53-null mouse mammary gland tumor model that closely mimics human breast cancer, we have identified, by limiting dilution transplantation and in vitro mammosphere assay, a Lin⁻CD29^HCD24^H subpopulation of tumor-initiating cells. Upon subsequent transplantation, this subpopulation generated heterogeneous tumors that displayed properties similar to the primary tumor. Analysis of biomarkers suggests the Lin⁻CD29^HCD24^H subpopulation may have arisen from a bipotent mammary progenitor. Differentially expressed genes in the Lin⁻CD29^HCD24^H mouse mammary gland tumor-initiating cell population include those involved in DNA damage response and repair, as well as genes involved in epigenetic regulation previously shown to be critical for stem cell self-renewal. These studies provide in vitro and in vivo data that support the cancer stem cell (CSC) hypothesis. Furthermore, this p53-null mouse mammary tumor model may allow us to identify new CSC markers and to test the functional importance of these markers. [Cancer Res 2008;68(12):4674–82]