The enrichment of cancer stem cell (CSC)-like cellular states has not previously been considered to be a causative mechanism in the generalized progression of EGFR-mutant non-small cell lung carcinomas (NSCLC) after an initial response to the EGFR tyrosine kinase inhibitor erlotinib. To explore this possibility, we utilized a pre-clinical model of acquired erlotinib resistance established by growing NSCLC cells containing a TKI-sensitizing EGFR exon 19 deletion (ΔE746-A750) in the continuous presence of high doses of erlotinib. Genome-wide analyses using Agilent 44K Whole Human Genome Arrays were evaluated via bioinformatics analyses through GSEA-based screening of the KEGG pathway database to identify the molecular circuitries that were over-represented in the transcriptomic signatures of erlotinib-refractory cells. The genomic spaces related to erlotinib resistance included a preponderance of cell cycle genes (E2F1, - 2, CDC2, -6) and DNA replication-related genes (MCM4, - 5, - 6, - 7), most of which are associated with early lung development and poor prognosis. In addition, metabolic genes such as ALDH1A3 (a candidate marker for lung cancer cells with CSC-like properties) were identified. Thus, we measured the proportion of erlotinib-resistant cells expressing very high levels of aldehyde dehydrogenase (ALDH) activity attributed to ALDH1/3 isoforms. Using flow cytometry and the ALDEFLUOR(®) reagent, we confirmed that erlotinib-refractory cell populations contained drastically higher percentages (> 4500%) of ALDH(bright) cells than the parental erlotinib-responsive cells. Notably, strong decreases in the percentages of ALDH(bright) cells were observed following incubation with silibinin, a bioactive flavonolignan that can circumvent erlotinib resistance in vivo. The number of lung cancer spheres was drastically suppressed by silibinin in a dose-dependent manner, thus confirming the ability of this agent to inhibit the self-renewal of erlotinib-refractory CSC-like cells. This report is the first to show that: (1) loss of responsiveness to erlotinib in EGFR-mutant NSCLC can be explained in terms of erlotinib-refractory ALDH(bright) cells, which have been shown to exhibit stem cell-like properties; and (2) erlotinib-refractory ALDH(bright) cells are sensitive to the natural agent silibinin. Our findings highlight the benefit of administration of silibinin in combination with EGFR TKIs to target CSCs and minimize the ability of tumor cells to escape cell death in EGFR-mutant NSCLC patients.