Recent studies using single-cell RNA sequencing technology have uncovered several subpopulations of CD4 T cells that accumulate with aging. These age-associated T cells are emerging as relevant players in the onset of inflammaging and tissue senescence. Here, based on information provided by single-cell RNA sequencing data, we present a flow cytometry panel that allows the identification of age-associated T cell subsets in systematic larger analysis in mice. We use this panel to evaluate at the single-cell level mitochondrial and senescence marks in the different age-associated CD4 T cell subpopulations. Our analysis identifies a subpopulation of regulatory T (T) cells that is characterized by the extracellular expression of the co-inhibitory molecule killer cell lectin-like receptor subfamily G member 1 (KLRG1) and accumulates with aging in humans and mice. KLRG1-expressing T cells display senescence features such as mitochondrial alterations, increased expression of cell-cycle regulators and genomic DNA damage. Functionally, KLRG1 T cells show a reduced suppressive activity in vivo accompanied by a pro-inflammatory phenotype.