Patients with lung tumors harboring activating mutations in the EGF receptor (EGFR) show good initial treatment responses to the EGFR tyrosine kinase inhibitors (TKIs) erlotinib or gefitinib. However, acquired resistance invariably develops. Applying a focused shRNA screening approach to identify genes whose knockdown can prevent and/or overcome acquired resistance to erlotinib in several EGFR-mutant non-small cell lung cancer (NSCLC) cell lines, we identified casein kinase 1 alpha (CSNK1A1, CK1 alpha). We found that CK1 alpha suppression inhibits the NF-kappaB pro-survival signaling pathway. Furthermore, down-regulation of NF-kappaB signaling by approaches independent of CK1 alpha knockdown can also attenuate acquired erlotinib resistance, supporting a role for activated NF-kappaB signaling in conferring acquired drug resistance. Importantly, CK1 alpha suppression prevented erlotinib resistance in an HCC827 xenograft model in vivo. Our findings suggest that patients with EGFR-mutant NSCLC might benefit from a combination of EGFR TKIs and CK1 alpha inhibition to prevent acquired drug resistance and prolong disease-free survival.