Conditional ablation of defined cell populations in vivo can be achieved using genetically engineered mice in which the human diphtheria toxin (DT) receptor (DTR) is placed under control of a murine tissue-specific promotor, such that delivery of diphtheria toxin selectively ablates cells expressing the high-affinity human DTR. Cells expressing only the endogenous low-affinity mouse DTR are assumed to be unaffected. Surprisingly, we found that systemic DT administration induced rapid regression of murine EGFR-mutant lung adenocarcinomas in the absence of a transgenic allele containing human DTR. DT enzymatic activity was required for tumor regression, and EGFR-mutant tumor cells were the primary targets of DT toxicity. In FVB mice, EGFR-mutant tumors upregulated expression of HB-EGF, which is the DTR in mice and humans. HB-EGF blockade with CRM197, an enzymatically inactive DT mutant, partially abrogated DT-induced tumor regression. These results suggest that elevated expression of murine HB-EGF (low-affinity DTR) confers sensitivity to DT in EGFR-mutant tumors, demonstrating a biological effect of DT in mice lacking transgenic DTR alleles and highlighting a unique vulnerability of EGFR-mutant lung cancers.