The major drawback with cancer therapy is the development of resistant cells within tumors due to their heterogeneous nature and due to inadequate drug delivery during chemotherapy. Therefore, the propagation of injury (“bystander effect” (BE)) from directly damaged cells to other cells may have great implications in cancer chemotherapy. The general advantage of the bystander cell killing phenomenon is the large therapeutic index that can be achieved. Experiments suggest that this phenomenon is detected in radiation therapy as well as in gene therapy in conjunction with chemotherapy. In the present study, we developed an original in vitro model dedicated to the exploration of bystander cytotoxicity induced during breast carcinoma chemotherapy. In brief, we investigated this perpetuation of injury on untreated bystander MCF‐7 breast cancer cells which were coplated with 5‐fluorouracil (5‐FU)‐treated MDA‐MB‐231 breast cancer cells. To achieve this goal, a specific in vitro coculture model which involved mixing of aggressive MDA‐MB‐231 breast cancer cells with enhanced green fluorescent protein (EGFP) expressing stable clone of non‐metastatic MCF‐7 breast cancer cells (MCF‐EGFP), was used. A bystander killing effect was observed in MCF‐EGFP cells cocultured with MDA‐MB‐231 cells pretreated with 5‐FU. The striking decrease in MCF‐EGFP cells, as detected by assaying for total GFP intensity, is mediated by activation of Fas/FasL system. The implication of Fas in MCF‐EGFP cell death was confirmed by using antagonistic anti‐FasL antibody that reverses bystander cell death by blocking FasL on MDA‐MB‐231 cells. In addition, inhibition of CD95/Fas receptor on the cell surface of MCF‐EGFP cells by treatment with Pifithrin‐alpha, a p53 specific transactivation inhibitor, partially abrogated the sensitivity of bystander MCF‐EGFP cells. Our data, therefore, demonstrates that the Fas/FasL system could be considered as a new determinant for chemotherapy‐induced bystander cell death in breast cancers. J. Cell. Biochem. J. Cell. Biochem. 101: 68–79, 2007. © 2007 Wiley‐Liss, Inc.