Fibroblasts are cells with a mesenchymal origin found in nearly every tissue. Their major function is to support tissue structure through the production and remodelling of the extracellular matrix (ECM), which is mainly composed by collagens. Moreover, fibroblasts influence tissue function by secreted cytokines and growth factors, mechanical forces and acting as a reservoir for tissue-specific mesenchymal cells. In the breast, fibroblasts are a major component of the tissue microenvironment, and women with high mammographic density (high collagen content in their breast ECM) have an increased risk of breast cancer. This suggests that fibroblast in healthy tissue can impact breast cancer development. Recent studies have described heterogeneity in cancer-associated fibroblasts (CAFs), but an in-depth study of fibroblast heterogeneity in healthy mammary gland is still missing. In this study, we aim to unravel the heterogeneity in the fibroblast compartment of the mammary gland, and to investigate their functions and their potential role in breast carcinogenesis. Using single-cell RNA-sequencing, we have uncovered four major subsets of mouse mammary fibroblasts and their contribution to key stages of mammary gland development. Similar to what has been recently shown for other tissues, we identified a population of ‘progenitor’ fibroblasts that serves as a reservoir for more specialized fibroblasts in breast tissue. We are currently establishing surface markers for prospective isolation of the different subsets and cell assays to test their function. Our work will reveal novel fibroblast populations as well as their markers and key pathways that maintain tissue homeostasis. In the future, we want to harness this knowledge to investigate these pathways in breast cancer.