Hence, a variety of approaches have been developed for quantifying and studying these specific mammary cell subsets. Here the authors describe how to perform a three dimensional ( 3D ) Mammary Colony-Forming Cell ( Ma-CFC ) Assay, an in vitro functional assay suitable for the detection and analysis of mammary progenitor cells in feeder-free culture conditions. The number and the cell composition of the resulting colonies will vary according to the frequency and the differentiation potential of the progenitors, respectively.