Krüpple-like factor 5 (KLF5) is required for the development of the embryo andmultiple organs, such as the lung and intestine. KLF5 plays a pro-proliferativeand oncogenic role in several carcinomas, including breast cancer. However, itsrole in normal mammary gland development and oncogenesis has not beenelucidated in vivo. In this study, we used mammary gland-specific Klf5conditional knockout mice derived by mating Klf5-LoxP and MMTV-Cre mice.The genetic ablation of Klf5 suppresses mammary gland ductal elongation andlobuloalveolar formation. Klf5 deficiency inhibits mammary epithelial cellproliferation, survival, and stem cell maintenance. Klf5 promotes mammarystemness, at least partially, by directly promoting the transcription of Slug. Finally,Klf5 depletion suppressed PyMT-induced mammary gland tumor cell stemness,tumor initiation and growth in vivo. Slug also mediated these functions of Klf5 invivo.