Characterization of the molecular pathways that are required for the viability and maintenance of self-renewing tumor-initiating cells may ultimately lead to improved therapies for cancer. In this study, we show that a CD133⁺/CD44⁺ population of cells enriched in prostate cancer progenitors (PCaPs) has tumor-initiating potential and that these progenitors can be expanded under nonadherent, serum-free, sphere-forming conditions. Cells grown under these conditions have increased in vitro clonogenic and in vivo tumorigenic potential. mRNA expression analysis of cells grown under sphere-forming conditions, compared with long-term monolayer cultures, revealed preferential activation of the PI3K/AKT signaling pathway. PI3K p110α and β-protein levels were higher in cells grown under sphere-forming conditions, and phosphatase and tensin homolog (PTEN) knockdown by shRNA led to an increase in sphere formation as well as increased clonogenic and tumorigenic potential. Similarly, shRNA knockdown of FoxO3a led to an increase in tumorigenic potential. Consistent with these results, inhibition of PI3K activity by the dual PI3K/mTOR inhibitor NVP-BEZ235 led to growth inhibition of PCaPs. Taken together, our data strongly suggest that the PTEN/PI3K/Akt pathways are critical for prostate cancer stem-like cell maintenance and that targeting PI3K signaling may be beneficial in prostate cancer treatment by eliminating prostate cancer stem-like cells.