Abstract The mammary gland consists of epithelial ducts that stretch out in a tree-like structure through a stromal fat pad. The ducts are formed by two layers, the inner layer composed of luminal cells and an outer layer of basal (myoepithelial) cells. Despite this relatively simple anatomy, the precise cellular composition and hierarchies are still ill-defined. Recent efforts to enrich, isolate, and characterize the different mammary epithelial cell compartments only used a handful of markers to define and trace cell populations. Therefore, there is a need for an unbiased and comprehensive description of mammary epithelial cells within the gland at different developmental stages. To this end we herein use single-cell RNA sequencing to determine the gene expression profile of individual cells across four adult developmental stages; nulliparous, mid-gestation, lactation, and post-weaning (full natural involution). Our data from over 23,000 individual cells identify distinct mammary epithelial cell populations and allow their hierarchical structure across development to be charted. The luminal compartment showed a differentiation structure with a common progenitor that can give rise to intermediate progenitors of both alveolar and hormone-sensing luminal cells. In contrast, we captured only few cells that were transitioning between basal and luminal cells, suggesting that the basal compartment is less involved in maintaining luminal cells in adult tissue homeostasis. Interestingly, the transcriptional profile of some cell types appeared to be more affected by gestation and lactation. For example, our analysis revealed a cluster of luminal progenitor cells in post-involution glands, which is distinct from progenitors found in nulliparous glands. The post-involution progenitor cells share all the luminal progenitor characteristics with their nulliparous counterparts but were marked by higher expression of genes involved in milk synthesis and the immune response, suggesting that these cells maintain a memory of having undergone a full pregnancy cycle. This is especially interesting in the light of the protective effect of early pregnancies on breast cancers. The data also showed that only few clusters could be fully characterized by a single marker gene. We argue instead that the epithelial cells—especially in the luminal compartment—should rather be conceptualized as being part of a continuous spectrum of differentiation. This view highlights the plasticity of the tissue and might help to explain some of the conflicting results from lineage tracing studies. Our work provides a foundation for understanding the cellular aspects of the developmental biology of the mammary gland. This is vital to understand the early steps of tumor development. We hope that our dataset can also be mined to help to gain insights into the cells-of-origin for various breast cancers. This abstract is also being presented as Poster A66. Citation Format: Karsten Bach, Sara Pensa, David J. Adams, John C. Marioni, Walid T. Khaled. Differentiation dynamics of the developing mammary gland revealed by single-cell RNA-sequencing [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr PR04.