Mesenchymal stromal cells (MSCs) are a heterogeneous population of non-hematopoietic adult stem cells derived from the embryonic mesoderm. They possess self-renewal and multipotent differentiation capabilities, allowing them to give rise to mesodermal cell types, such as osteoblasts, chondroblasts, and adipocytes, as well as non-mesodermal cells, including neuron-like cells and endothelial cells. MSCs play a vital role in maintaining homeostasis across various tissues by facilitating tissue repair, immune regulation, and inflammatory response balance. Initially identified in bone marrow, MSCs have since been found in multiple tissues, including muscle, adipose tissue, and dental pulp, and are characterized by specific surface markers and differentiation abilities.Aging induces cellular senescence, an irreversible growth arrest linked to various stressors, which has significant implications for regenerative medicine. While initially viewed as a protective mechanism against tumorigenesis, the accumulation of senescent cells, particularly in MSCs, leads to age-related diseases through the senescence-associated secretory phenotype (SASP). The onset of senescence in MSCs diminishes their therapeutic potential and contributes to homeostatic imbalance. Key drivers of MSC senescence include genetic damage, noncoding RNA, and mitochondrial dysfunction, among others.This study outlines the principal methodologies for the isolation and characterization of MSCs, alongside techniques to induce acute senescence via hydrogen peroxide or irradiation, as well as replicative senescence, to investigate senescence-related changes in vitro. Understanding the mechanisms of MSC senescence will provide critical insights into the molecular pathways of aging and pave way for advancements in cellular therapies targeting age-related diseases.