Lung cancer is a leading cause of cancer-related death worldwide. Every year it claims more lives than other common malignancies, breast, prostate, and colorectal cancers, combined. And despite recent improvements in disease diagnostics and treatment, drug resistance and tumor relapse remain major contributors to low patient survival. Both issues have been largely attributed to the existence of cancer stem-like cells (CSCs) or tumor initiating cells (TICs). CSCs are a small cell population within heterogeneous tumors that is inherently resistant to apoptosis, chemo- and radiation therapy. Thus, it is critical to elucidate CSC-specific signaling pathways and identify early biomarkers for therapeutic intervention. This body of work investigates molecular mechanisms of CSC regulation by an embryonic transcription factor SOX9. We show here that SOX9 is upregulated upon exposure to cisplatin and positively regulates cancer stem-like properties and chemoresistance of non-small cell lung cancer (NSCLC) cells. Moreover, we demonstrate for the first time that the stem-cell marker ALDH1A1 is a direct SOX9 transcriptional target. We also show that SOX9 expression is upregulated in human lung epithelial cells chronically exposed to single-walled carbon nanotubes (SWCNT). SOX9 was identified as a critical factor promoting metastatic and stem-like properties in these cells. We also observed a correlation between SOX9 levels and ALDH expression and activity in this model of carcinogenesis, suggesting that activation of SOX9-ALDH axis may have a broader role in the regulation of cellular stress responses. Taken together, our novel findings on the role of SOX9-ALDH axis support the use of this CSC regulator as a prognostic marker of cancer chemoresistance and as a potential drug target for CSC therapy. The SOX9-ALDH axis could also have a broad role in the regulation of CSC survival under different stress conditions.