Normal human mammary tissue is composed of a glandular epithelium embedded within a fibrous and fatty stroma. Collagenase and hyaluronidase digestion of normal reduction mammoplasty specimens followed by differential centrifugation yields a suspension of single cells and cell aggregates that contain elements of the terminal ductal lobular units and stromal components of the mammary gland. The terminal ductal lobular units (TDLU) can be further dissociated to complete viable single-cell suspensions by treatment with trypsin, dispase II, and deoxyribonuclease I. These suspensions are suitable for cell separation and analysis in culture. Such studies indicate the existence of biologically distinct subpopulations of luminal-restricted, myoepithelial-restricted, and bipotent mammary epithelial progenitors detected by their ability to generate colonies of the corresponding progeny types in serum-free cultures. This review summarizes the methodology of the techniques required to generate and characterize the colonies obtained in vitro from these progenitors, as well as the special considerations and potential pitfalls associated with performing these protocols.