The mammary epithelium comprises basal and luminal cell layers, with the basal compartment harboring stem/progenitor cells that can exhibit bipotent potential to generate both myoepithelial and luminal lineages under appropriate conditions. We present a robust two-dimensional (2D) culture protocol for the long-term expansion of primary human mammary basal cells using F medium supplemented with a defined chemical cocktail, “YDAC.” This system preserves basal cell identity in commercially available primary human mammary epithelial cells (HMECs) during extended culture, providing a renewable source of undifferentiated basal cells. Upon YDAC withdrawal, the expanded 2D basal cells undergo synchronized epithelial differentiation, accompanied by lineage marker shifts and characteristic changes in Claudin expression. Prolonged culture in YDAC-containing medium beyond confluence induces the formation of bilayered 3D epithelial structures, while embedding in Matrigel yields heterogeneous organoids. This combination of scalable basal cell expansion and controllable differentiation enables comprehensive molecular and functional analyses. It also facilitates integration of 2D and 3D epithelial models to ensure reproducible, physiologically relevant insights.