Mammary gland stem cells are a quiescent and self-renewing population within the mammary gland that are capable of giving rise to the differentiated ductal, alveolar and myoepithelial cells. To identify mammary gland stem cells, several investigators have employed a variety of methods including: non-adherent mammosphere cultures; 5-bromo-2-deoxy-uridine (BrdU) label-retention studies; cell-surface markers, such as Sca1 and CD49f; and Hoechst dye efflux. These methods have helped identify and further characterize signal transduction pathways such as the Notch, Wnt and Hedgehog pathways that may be important for the self-renewal and fate determination of mammary gland stem cells. Stem cells within the mammary gland have been proposed to underpin many types of breast cancer. A better understanding of the signal transduction pathways and the molecules that are responsible for the self-renewal and survival of these cells will be essential in the design of more effective therapies aimed at the eradication of both cancer-initiating cells and breast cancer stem cells.