The regulation checkpoints of CD4CD25 expansion in presence and absence of transcription factor FOXP3 are critical to maintain balance between inflammatory TH17 and anti-inflammatory FOXP3CD4CD25 Treg cells. We investigated impact of potent inflammatory mediator nitric oxide NO(.) on commitment of normal spleen T cells following activation. The single cell suspension of healthy C57BL/6 mice splenic CD4 cells was stimulated by plate-bound antiCD3/antiCD28 antibodies in presence of cytokine cocktail IL2, IL6, TGFβ and anti-IL4, anti-IFN γ antibodies with and without GSNO for 72h. The results showed that incubation with GSNO reduced number of activated CD4CD25 cells. We found decreased level of CD4FOXP3 than CD4RORγt cells in presence of GSNO. The AnnexinV-propidium iodide staining of CD4CD25 cells followed by flow cytometry suggested presence of GSNO increased apoptosis of CD4+CD25+ but not CD4+CD25-cells.