Selective killing of tumor cells is an important goal for cancer therapeutics. The tumor suppressor transcription factor p53 is absent or mutated in more than 50% of human tumors. Thus, determining approaches that use p53 status to regulate therapy may be an important strategy for attaining cancer selectivity. We have shown previously that a designed transcriptional repressor, K2-5F, strongly and selectively reduces the expression of its target gene MDR1. In this study, we exploited p53 status and the strong repressor activity of K2-5F to establish a system for preferential killing of p53-negative cells. In this system, the expression of K2-5F is induced by p53 in normal cells, and the K2-5F repressor then inhibits the expression of herpes simplex virus thymidine kinase (HSV-TK) driven by an MDR1 minipromoter. In p53-deficient cells, little K2-5F is expressed, and thus HSV-TK is expressed, allowing the cells to be killed by ganciclovir (GCV). K2-5F induced by exogenous p53 dramatically reduced the expression of HSV-TK in human embryonic kidney 293 cells, and it subsequently increased cell survival in response to GCV. To further evaluate this approach in a uniform genetic background, we developed Saos-2 cells stably expressing physiological levels of p53 and paired them with wild-type p53-negative Saos-2 cells. Stable expression of moderate levels of p53 in Saos-2 cells was able to induce the expression of K2-5F and reduce HSV-TK expression and resulted in a modest but distinct protection from GCV toxicity. Thus, this system may be suitable for further development as an approach to selective cancer therapy.