Abstract Basal-like breast cancers (BLBC) are heterogenous, poorly differentiated and present with limited therapeutic opportunities and a poor prognosis. We have investigated the role of the HLH transcription factor ID4 in breast stem cells and basal breast cancer aetiology. Using an Id4GFP knock-in mouse and single cell gene expression studies we show that ID4 defines a subset of Cd24+Cd29hi basal mammary epithelial cells enriched for stem cell gene expression and mammary repopulating capacity. Homozygous deletion of Id4 followed by mammary transplantation reveals a cell-autonomous requirement for Id4 in mammary ductal morphogenesis in vivo. shRNA-mediated depletion of Id4 in the Comma-D mammary stem cell line in vitro shows that Id4 is required for epithelial cell proliferation. Conversely, over-expression of Id4 maintains the stem cell pool by restricting luminal differentiation of Comma-D cells. This effect of Id4 is mediated via suppression of BrCa1 expression and the Notch-Elf5 axis. In clinical breast cancer specimens, ID4 expression is absent in luminal tumours but very high in approximately half of all BLBCs, a proportion of which possess ID4 gene amplification. These tumours are characterized by unique transcriptomes with many features of mammary stem cells. Furthermore, ID4-positive BLBCs have a very poor prognosis across multiple independent tissue cohorts. This study identifies ID4 as a master regulator of mammary stem cell homeostasis and suggests that a subset of aggressive BLBCs derive from a primitive mammary stem or progenitor cell with basal characteristics. Citation Format: Simon Junankar, Laura Baker, Kyuson Yun, Alexander Swarbrick. Inhibitor of differentiation 4 (ID4) maintains mammary stem cell homeostasis and identifies a poor-prognosis subset of basal-like breast cancers with a putative stem cell of origin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-62. doi:10.1158/1538-7445.AM2014-LB-62