Maternal embryonic leucine zipper kinase (MELK) is expressed in several developing tissues, in the adult germ line, and in adult neural progenitors. MELK expression is elevated in aggressive undifferentiated tumors, correlating with poor patient outcome in human breast cancer. To investigate the role of MELK in mammary tumorigenesis in vivo, we used a MELK-green fluorescent protein (GFP) reporter mouse, which allows prospective isolation of MELK-expressing cells based on GFP fluorescence. We found that in the normal mammary gland, cells expressing high levels of MELK were enriched in proliferating cells that express markers of mammary progenitors. The isolation of cells with high levels of MELK in mammary tumors from MMTV-Wnt1/MELK-GFP bitransgenic mice resulted in a significant enrichment of tumorsphere formation in culture and tumor initiation after transplantation into mammary fat pads of syngeneic mice. Furthermore, using lentiviral delivery of MELK-specific shRNA and limiting dilution cell transplantations, we showed that MELK function is required for mammary tumorigenesis in vivo. Our findings identify MELK as a potential target in breast tumor-initiating cells. Cancer Res; 70(21); 8863–73. ©2010 AACR.