Breast milk is chock-full of nutrients, immunological factors, and cells that aid infant development. Maternal cells are the least studied breast milk component, and their unique properties are difficult to identify using traditional techniques. Here, we characterized the cells in mature-stage breast milk from healthy donors at the protein, gene, and transcriptome levels. Holistic analysis of flow cytometry, quantitative polymerase chain reaction, and single-cell RNA sequencing data identified the predominant cell population as epithelial with smaller populations of macrophages and T cells. Two percent of epithelial cells expressed four stem cell markers: SOX2, TRA-1-60, NANOG, and SSEA4. Furthermore, milk contained six distinct epithelial lactocyte subpopulations, including three previously unidentified subpopulations programmed toward mucosal defense and intestinal development. Pseudotime analysis delineated the differentiation pathways of epithelial progenitors. Together, these data define healthy human maternal breast milk cells and provide a basis for their application in maternal and infant medicine.