Dramatic changes in gene expression are required for a stem cell to undergo differentiation into a specialised cell. Transcription factors and chromatin remodellers are central in determining gene expression profiles and hence cell identity during development, and these programs are often deregulated during tumorigenesis. The mammary gland is a particularly useful model to study such regulatory programs due to its postnatal development. Our lab and others have demonstrated that the transcription factor ID4 is a master regulator of stem/progenitor cells in the mammary gland where it supresses key pathways critical for luminal cell differentiation including ER, Elf5, Notch and Brca11,2. We have also shown that ID4 is overexpressed and required by a subset of poorly differentiated basal-like breast cancers1. This project aims to define the molecular and cellular mechanisms by which ID4 controls mammary epithelial fate. ID4 lacks a DNA binding domain, and is thought to function by interacting with other transcription factors. We have used a novel technique called Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins (RIME) to firstly identify these factors, and secondly to investigate how they function. We have found that ID4 binds to E-proteins. These transcription factors are pivotal to many developmental systems but have uncharacterised roles in mammary gland lineage commitment. The E-protein HEB has high expression in virgin mammary epithelium and interacts with a suite of epigenetic regulators, including DNA and histone modifying enzymes. Some of these epigenetic regulators have recently been implicated in mammary gland development and breast cancer. We hypothesise that ID4 regulates the activity of E-proteins, which function through modulation of the epigenome to control cellular homeostasis and differentiation. Consistent with this hypothesis, differential gene expression performed in sorted mammary subpopulations from ID4 germ line knockout mice reveals regulation of key developmental pathways. Studying the mechanism of ID4 function has revealed novel insights into the transcriptional and epigenetic complexes regulating cell fate and lineage commitment in the mammary epithelium.