Cancer stem-like cells (CSC) are thought to drive tumor initiation, metastasis, relapse, and therapeutic resistance, but their specific pathogenic characters in many cancers, including non-small cell lung cancer (NSCLC), have yet to be well defined. Here, we develop findings that the growth factor HGF promotes CSC sphere formation in NSCLC cell populations. In patient-derived sphere-forming assays (PD-SFA) with HGF, CD49f and CD104 were defined as novel markers of lung CSC (LCSC). In particular, we isolated a subpopulation of CD166CD49fCD104Lin LCSC present in all human specimens of NSCLC examined, regardless of their histologic subtypes or genetic driver mutations. This specific cell population was tumorigenic and capable of self-renewal, giving rise to tumor spheres and orthotopic lung tumors in immune-compromised mice. Mechanistic investigations established that was preferentially expressed in this cell subpopulation and required for self-renewal via the transcription factor HES1. Through a distinct HES1-independent pathway, NOTCH1 also protected LCSCs from cisplatin-induced cell death. Notably, treatment with a γ-secretase inhibitor that blunts NOTCH1 function ablated self-renewing LCSC activity and restored platinum sensitivity and Overall, our results define the pathogenic characters of a cancer stem-like subpopulation in lung cancer, the targeting of which may relieve platinum resistance in this disease. .