Abstract The mechanisms of lung cancer evading the immune system are still not fully understood. As regulatory T cells (Tregs) exhibit a pro-tumoral function they are in focus of immunotherapies. Peripheral Treg differentiation is driven by TCR and its co-stimulation. A further crucial signal displays the binding of IL-2 to the high affinity receptor CD25 with downstream involvement of Signal Transducer and Activator of Transcription 5 (STAT5). STAT5 activation as well as TGF-beta stimulation in the periphery leads to expression of the Treg key factor FoxP3. In this study, we wanted to investigate the role of STAT5 and its activated status via phosphorylation (pSTAT5) within lung cancer. We thus isolated peripheral blood mononuclear cells (PBMC) from NSCLC patients and healthy donors. PBMCs were cultured under different stimulation conditions for 4 days and analyzed by FACS. Here we found CD4+ T cells with high induction of pSTAT5A and CD25 upon aCD3 and aCD28 antibody treatment. Moreover, CD4+CD25+FoxP3+ Tregs were detected under T cell activating conditions, and were found to be increased by TGFβ in smokers and NSCLC patients. The Treg population was further inducible by adding low doses of IL-2 while CD4+CD25+FoxP3− effector T cells decreased in the presence of TGFβ. Our findings point to an immunosuppressive microenvironment induced by the tumor with increased TGFβ and reduced IL-2 forcing effector T cells into a Treg state. These preliminary data suggest that current immunotherapy by aCD3/aCD28 antibody treatment could be improved by an additional blockade of TGFβ and induction of IL-2 levels. This treatment would result in an increase of CD4+CD25+pSTAT5+FoxP3− effector cells and a reduction of CD4+CD25+FoxP3+STAT5+ Tregs.