Metastasis is the main reason for lung cancer related mortality but little is known about specific determinants of successful dissemination from primary tumors and metastasis initiation. Here we show that CD133+/CXCR4+ cancer initiating cells (CICs) directly isolated from patient-derived xenografts (PDX) of non-small cell lung cancer (NSCLC) are endowed with superior ability to seed and initiate metastasis at distant organs. We additionally report that CXCR4 inhibition successfully prevents the increase of cisplatin-resistant CD133+/CXCR4+ cells in residual tumors and their metastatization. Immunophenotypic analysis of lung tumor cells intravenously injected or spontaneously disseminated to murine lungs, demonstrates the survival advantage and increased colonization ability of a specific subset of CD133+/CXCR4+ with reduced expression of epithelial cell adhesion molecule (EpCAM-), that also shows the greatest in vitro invasive potential. We next prove that recovered disseminated cells from lungs of PDX bearing mice enriched for CD133+/CXCR4+/EpCAM- CICs are highly tumorigenic and metastatic. Importantly, microenvironment stimuli eliciting EMT, including signals from cancer-associated fibroblasts, are able to increase the dissemination potential of lung cancer cells through the generation of CD133+/CXCR4+/EpCAM- subset. These findings also have correlates in patient samples where disseminating CICs are enriched in metastatic lymphnodes (20-fold, p=0.006) and their detection in primary tumors is correlated with poor clinical outcome (DFS p=0.03; OS p=0.05). Overall these results highlight the importance of specific cellular subsets in the metastatic process, the need for in depth characterization of disseminating tumor cells and the potential of therapeutic strategies targeting both primary tumor and tumor-microenvironment interactions.