Gene pairs specific for a toxin and its antitoxin are called toxin-antitoxin modules and are found on the chromosomes of many bacteria. The most studied of these modules is Escherichia coli mazEF, in which mazF encodes a stable toxin, MazF, and mazE encodes a labile antitoxin, MazE, which prevents the lethal effect of MazF. In a previous report from this laboratory, it was shown that mazEF-mediated cell death is a population phenomenon requiring a quorum-sensing peptide called the extracellular death factor (EDF). EDF is the linear pentapeptide NNWNN (32). Here, we further confirm that EDF is a signal molecule in a mixed population. In addition, we characterize some physiological conditions and genes required for EDF production and response. Furthermore, stress response and the gene specifying MazEF, the Zwf (glucose-6-phosphate dehydrogenase) gene, and the protease ClpXP are critical in EDF production. Significant strain differences in EDF production and response explain variations in the induction of mazEF-mediated cell death.