Transcriptional regulatory networks play a fundamental role in the determination of stem cell fate. However, relatively little is known about the transcription factors involved in mammary stem cell (MaSC) biology. Our lab and others have shown that Id4 marks a stem/progenitor cell in the MaSC-enriched basal compartment where it supresses key pathways critical for luminal cell differentiation including ER, Elf5, Notch and Brca11,2. We have also demonstrated that Id4 is expressed and required by a subset of aggressive basal-like breast cancers (BLBC) which are transcriptionally similar to the MaSC-enriched basal population in the normal mammary gland1. This project aims to unravel the molecular and cellular functions of Id4 in mammary gland development. Using a novel EGFP knock-in reporter mouse allows us to isolate Id4-positive mammary cells by Fluorescence Activated Cell Sorting (FACS) and characterise the function of these cells by transplantation assays and the transcriptome of these cells by RNA-Sequencing. Transplantation assays have revealed that Id4-positive cells are better able to reconstitute cleared mammary fat pads of naïve wildtype recipients than Id4-negative cells, demonstrating higher cell intrinsic stem cell activity1. Id transcription factors are conventionally thought to function by sequestration of bHLH transcription factors, the identity of which is unknown in mammary epithelium. To discover Id4 binding partners we are using a technique called Rapid Immunoprecipitation Mass Spectrometry of Endogenous Proteins (RIME) in the normal mammary cell line, Comma Dβ. Using this technique we have identified and validated several transcription factor binding partners which we are currently investigating. Studying the mechanism of Id4 function has revealed novel insights into the transcriptional complexes regulating cell fate and lineage commitment in the mammary epithelium.