Clinical breast cancer progresses via a multi-step carcinogenic process wherein genetic, molecular, endocrine and dietary factors play significant roles in the pathogenesis, prevention and therapy of the disease. Preclinical cell culture models, expressing clinically relevant genetic and endocrine defects and exhibiting quantifiable cancer risk, may provide facile, clinically translatable approaches to identify molecular targets and susceptible mechanistic pathways for the efficacy of novel interventional approaches. This review summarizes laboratory investigations focused on i) developing murine and human mammary tissue-derived cell culture models; ii) optimizing mechanism-based quantitative endpoint biomarker assays specific for carcinogenic risk and preventive/therapeutic efficacy; and iii) providing quantifiable proof-of-principle evidence for validation of the present cell culture approaches, capable of prioritizing efficacious lead compounds for subsequent in vivo animal studies and clinical trials for the prevention/therapy of breast cancer. Epithelial cell culture models are developed and characterized where the carcinogenic process is initiated by the targeted expression of clinically relevant oncogenes. The cell culture systems from mouse mammary tissue are in vitro approaches that complement the Ras and Myc transgenic mouse models. The human mammary tissue-derived systems are in vitro models for chemoendocrine, therapy-resistant, clinical, pre-invasive ER(-)/PR(-)/HER-2(+) comedo ductal carcinoma in situ, ER(+)/PR(+) chemoendocrine therapy-responsive breast cancer and ER(-)/PR(-)/HER-2(-) triple-negative chemoendocrine, therapy-resistant breast cancer. The oncogene-initiated phenotypes exhibit loss of homeostatic growth control, downregulation of cell apoptosis and gain of carcinogenic risk in vitro, as well as transplantable tumor development in vivo. Numerous mechanistically distinct, synthetic pharmacological agents, as well as naturally occurring dietary compounds, re-establish homeostatic growth control via cell cycle arrest and/or induction of cell apoptosis, downregulate oncogene-mediated cell signaling pathways, modulate the expression of numerous cell cycle regulatory and apoptosis-specific proteins and reduce carcinogenic risk in pre-neoplastic and carcinoma-derived cell culture models. These data validate the present cell culture approaches as novel, mechanism-based screens to evaluate and prioritize promising lead compounds for the prevention/therapy of clinical breast cancer.