Therapy resistance is one of the major impediments to successful cancer treatment. In breast cancer, a small subpopulation of cells with stem cell features, named breast cancer stem cells (BCSC), is responsible for metastasis and recurrence of the tumor. BCSC have the unique ability to grow under non-adherent conditions in "mammospheres". To prevent breast cancer recurrence and metastasis it will be crucial to eradicate BCSC.We used shRNA genetic screening to identify genes that upon knockdown enhance mammosphere formation in breast cancer cells. By integration of these results with gene expression profiles of mammospheres and NOTCH-activated cells, we identified FOXO3A. Modulation of FOXO3A activity results in a change in mammosphere formation, expression of mammary stem cell markers and breast cancer initiating potential. Importantly, lack of FOXO3A expression in breast cancer patients is associated with increased recurrence rate. Our findings provide evidence for a role for FOXO3A downstream of NOTCH and AKT that may have implications for therapies targeting BCSCs.