Cancer stem-like cells contribute to tumor heterogeneity and have been implicated in disease relapse and drug resistance. Here we show the coexistence of distinct breast cancer stem-like cells (BCSC) as identified by ALDH⁺ and CD29^hiCD61⁺ markers, respectively, in murine models of breast cancer. While both BCSC exhibit enhanced tumor-initiating potential, CD29^hiCD61⁺ BCSC displayed increased invasive abilities and higher expression of epithelial-to-mesenchymal transition and mammary stem cell–associated genes, whereas ALDH⁺ BCSC were more closely associated with luminal progenitors. Attenuating the autophagy regulator FIP200 diminished the tumor-initiating properties of both ALDH⁺ and CD29^hiCD61⁺ BCSC, as achieved by impairing either the Stat3 or TGFβ/Smad pathways, respectively. Furthermore, combining the Stat3 inhibitor Stattic and the Tgfβ-R1 inhibitor LY-2157299 inhibited the formation of both epithelial and mesenchymal BCSC colonies. In vivo, this combination treatment was sufficient to limit tumor growth and reduce BCSC number. Overall, our findings reveal a differential dependence of heterogeneous BCSC populations on divergent signaling pathways, with implications on how to tailor drug combinations to improve therapeutic efficacy. Cancer Res; 76(11); 3397–410. ©2016 AACR.