Prospective isolation and in vitro and in vivo analysis of primary mouse mammary epithelial cells has been used to separate cell subpopulations and identify stem, progenitor and differentiated cell compartments. Progress has been made from cell separation strategies based on a single marker of the luminal epithelial or myoepithelial compartments to use of markers that allow simultaneous isolation of non-epithelial, basal/myoepithelial and luminal epithelial cells. Transplant analysis has shown that mammary stem cells are found in the basal/myoepithelial compartment, whereas in vitro colony progenitors are found in the luminal compartment. A basal population enriched for stem cell activity can be purified from the myoepithelial cells and the most recent data shows that the luminal population can now be prospectively split into estrogen receptor positive and estrogen receptor negative cells. Future work aims to molecularly characterise these populations to identify new drug targets, which can be used to specifically kill breast cancer stem cells.