EGFR mutant lung cancers eventually become resistant to treatment with EGFR tyrosine kinase inhibitors (TKIs) such as erlotinib. The combination of an irreversible EGFR TKI, afatinib, and the anti-EGFR monoclonal antibody, cetuximab, depletes both phosphorylated and total EGFR and can overcome resistance in mouse lung tumor models and human patients with acquired resistance. Since afatinib is also a potent HER2 inhibitor, here we investigated the role of HER2 in EGFR mutant tumor cells. We show in vitro and in vivo that afatinib plus cetuximab or the related antibody, panitumumab, significantly inhibits HER2 phosphorylation. HER2 overexpression or knockdown confers resistance or sensitivity, respectively, in all studied cell line models. Fluorescent in situ hybridization analysis revealed that HER2 was amplified in 3 of 26 (12%) tumors with acquired resistance versus only 1 of 99 (1%) untreated lung adenocarcinomas (p=0.03 Fisher's exact test). Notably, HER2 amplification and EGFR T790M were mutually exclusive (0 of 17 T790M-positive versus 3 of 9 T790M-negative cases (p=0.02 Fisher's exact test)). Collectively, these results reveal a previously unrecognized mechanism of resistance to EGFR TKIs and provide a rationale to assess the status and possibly target HER2 in EGFR mutant tumors with acquired resistance to EGFR TKIs.