The hypothesis of cancer stem cells has been proposed to explain the therapeutic failure in a variety of cancers including lung cancers. Previously, we demonstrated acquisition of epithelial-mesenchymal transition, a feature highly reminiscent of cancer stem-like cells, in gefitinib-resistant A549 cells (A549/GR). Here, we show that A549/GR cells contain a high proportion of CXCR4+ cells that are responsible for having high potential of self-renewal activity in vitro and tumorigenicity in vivo. A549/GR cells exhibited strong sphere-forming activity and high CXCR4 expression and SDF-1α secretion compared with parent cells. Pharmacological inhibition (AMD3100) and/or siRNA transfection targeting CXCR4 significantly suppressed sphere-forming activity in A549 and A549/GR cells, and in various non-small cell lung cancer (NSCLC) cell lines. A549/GR cells showed enhanced Akt, mTOR and STAT3 (Y705) phosphorylation. Pharmacological inhibition of phosphatidyl inositol 3-kinase or transfection with wild-type PTEN suppressed phosphorylation of Akt, mTOR and STAT3 (Y705), sphere formation, and CXCR4 expression in A549/GR cells, whereas mutant PTEN enhanced these events. Inhibition of STAT3 by WP1066 or siSTAT3 significantly suppressed the sphere formation, but not CXCR4 expression, indicating that STAT3 is a downstream effector of CXCR4-mediated signaling. FACS-sorted CXCR4+ A549/GR cells formed many large spheres, had self-renewal capacity, demonstrated radiation resistance in vitro and exhibited stronger tumorigenic potential in vivo than CXCR4- cells. Lentiviral-transduction of CXCR4 enhanced sphere formation and tumorigenicity in H460 and A549 cells, whereas introduction of siCXCR4 suppressed these activities in A549/GR cells. Our data indicate that CXCR4+ NSCLC cells are strong candidates for tumorigenic stem-like cancer cells that maintain stemness through a CXCR4-medated STAT3 pathway and provide a potential therapeutic target for eliminating these malignant cells in NSCLC.