Abstract Introduction Cancer stem cells (CSCs) are implicated in tumor initiation and development of metastasis. However, whether CSCs also affect the immune system is not fully understood. We investigated correlations between the PD-L1⁺ CSCs, changes in T-cell phenotype in metastatic and non-metastatic lymph nodes (LNs) and response to treatment. Methods LNs’ aspirates were obtained during the EBUS/TBNA procedure of 20 NSCLC patients at different stages of the disease. CSCs and T-cell characteristics were determined by flow cytometry. Results PD-L1⁺ CSCs positively correlated with the percentage of Tregs, PD-1⁺ CD4 T cells and Tim3⁺ CD4⁺ T cells, whereas PD-L1⁺ CSCs were negatively correlated with CD4⁺ T cells and CD28⁺ CD4⁺ T cells. The percentage of PD-L1⁺ CSCs was higher in patients with progressive disease (PD) as compared to patients with stable disease (SD) or partial response (PR). Among T cells, only PD-1⁺ CD4⁺ T cells and Tim3⁺ CD4⁺ T-cell frequencies were higher in patients with PD as compared to patients with SD or PR. Conclusion The frequency of PD-L1⁺ CSCs associates with an altered T-cell frequency and phenotype indicating that CSCs can affect the immune system. The higher percentage of PD-L1⁺ CSCs in patients with PD may confirm their resistance to conventional therapy, suggesting that CSCs may be an interesting target for immunotherapy.