Abstract The dissemination of metastases from the primary tumor accounts for over 90% of cancer-associated mortality. Despite advances on treatment modalities involving chemotherapeutic drugs, radiotherapy, and surgery, metastasis almost invariably arises by persistence of unresolved cancer cells following initial treatment. One intriguing explanation for this resiliency is the cancer stem-cell (CSC) hypothesis. According to the CSC model, phenotypic heterogeneity exists within the tumor, and a minority fraction of cells are able to reconstitute the tumor following ablation by current treatments. In like manner to their “normal” stem-cell counterparts, CSCs are capable of unlimited self-renewal, and the ability to “differentiate” to reconstitute the phenotypic heterogeneity of the initial tumor. Recent studies have sought to identify cancer stem cells in numerous types of solid tumors, including breast and prostate. Among the cell surface antigens utilized to identify the CSC subpopulations are CD24, CD44, CD133, and other factors associated with epithelial-to-mesenchymal transition (EMT). In this study, we sought to investigate the phenotypic changes associated with cell-cycle in breast (BCa-SC) and prostate (PCa-SC) cancer. We characterized in vitro the malignant and osteolytic human breast cancer cell line MDA-MB-231 and human prostate cancer cell line PC3, with the goal of identifying an optimal checkpoint where the CSC subpopulation would be most vulnerable to subsequent targeted chemotherapy. We found that common CSC markers are expressed in a time-dependent manner due to changes in the cell cycle. Our hypothesis is that temporal inhibition of the cell cycle when the CSC phenotype is least prominent will circumvent the chemo-resistance inherent to CSC. Citation Format: Joseph Feduska, Carnellia Lee, Selvarangan Ponnazhagan. Cell cycle regulation influences the phenotype of breast and prostate cancer stem cells in vitro. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2539.