Lung cancer is the leading cause of cancer-related death in the United States and worldwide. Lung cancers that are driven by mutations in the Epidermal Growth Factor Receptor (EGFR) are treated with targeted anti-EGFR therapy, such as first-generation tyrosine kinase inhibitors (TKIs) erlotinib or gefitinib. Unfortunately, all patients whose tumors initially respond to erlotinib or gefitinib will eventually go on to develop primary acquired resistance, most commonly through a second-site EGFR mutation, T790M. The combination of a second-generation EGFR TKI, afatinib, plus the anti-EGFR antibody, cetuximab, may overcome primary acquired resistance. Unfortunately, secondary acquired resistance is also seen with the new drug combination, and the underlying mechanisms have not yet been determined. Using tumor sample from a patient with secondary acquired resistance, we identified two unexpected NF2 mutations, neither of which was detected in the patient’s pre-treatment sample, suggesting that mutations in NF2 were acquired during treatment. The NF2 gene encodes merlin, a known tumor suppressor that is thought to regulate EGFR. Using cell line and animal models, we demonstrate that loss of NF2 is sufficient to induce resistance to TKI in EGFR-mutant cells. Co-treatment with the mTORC1 inhibitor, rapamycin, re-sensitizes cells to TKI. These studies suggest a novel potential mechanism of acquired resistance to targeted therapy in EGFR-mutant lung cancer. To further explore acquired resistance, we also evaluated a novel, irreversible, mutant-specific third-generation EGFR TKI, AZD9291. Pre-clinically, this drug potently inhibits signaling pathways and cellular growth in both EGFR-mutant and EGFR-mutant/T790M mutant cell lines in vitro, with lower activity against wild-type EGFR lines, translating into profound and sustained tumor regression in EGFR mutant tumor xenograft and transgenic models. We use cell line and mouse models to characterize AZD9291, providing the foundation for a recently-initiated clinical trial. In sum, this work evaluates mechanisms of acquired resistance to targeted therapy in EGFR-mutant lung cancer and identifies potential strategies to overcome such resistance.