The p53 tumor suppressor protein plays a crucial role in influencing cell fate decisions in response to cellular stress. As p53 elicits cell cycle arrest, senescence or apoptosis, the integrity of the p53 pathway is considered a key determinant of anti-tumor responses. p53 can also promote autophagy, however the role of p53-dependent autophagy in chemosensitivity is poorly understood. VMY-1-103 (VMY), a dansylated analog of purvalanol B, displays rapid and potent anti-tumor activities, however the pathways by which VMY works are not fully defined. Using established prostate cancer cell lines and novel conditionally reprogrammed cells (CRCs) derived from prostate cancer patients; we have defined the mechanisms of VMY-induced prostate cancer cell death. Herein, we show that the cytotoxic effects of VMY required a p53-dependent induction of autophagy, and that inhibition of autophagy abrogated VMY-induced cell death. Cancer cell lines harboring p53 missense mutations evaded VMY toxicity and treatment with a small molecule compound that restores p53 activity re-established VMY-induced cell death. The elucidation of the molecular mechanisms governing VMY-dependent cell death in cell lines, and importantly in CRCs, provides the rationale for clinical studies of VMY, alone or in combination with p53 reactivating compounds, in human prostate cancer.