Mesenchymal stem cells (MSCs) are attracting a great deal of attention because they represent a valuable source of cells for use in regenerative medicine. In human cell culture it is important to obtain large numbers of cells for use in therapy. In this study, we attempted to prolong life span of a marrow-derived mesenchymal stem cell using a combination of growth factors and hormones. Furthermore we tested whether chemically defined culture conditions are sufficient for maintenance of multipotent mesenchymal stem cells. Epidermal growth factor, platelet-derived growth factor-BB (PDGF-BB), acidic fibroblast growth factor (FGF), basic FGF, and leukemia inhibitory factor were found to be key factors for the mesenchymal stem cell proliferation. The combination of these growth factors showed extremely strong mitogenic activity, and simultaneously induced the expression of cyclin-dependent kinase inhibitor p16ink4a protein and premature senescence more rapidly than serum-supported culture conditions. The induction of p16ink4a by growth factors was mediated through the mitogen-activated protein kinase (MAPK) cascade. Excess growth stimulation by growth factors was thus one of the culture stress signals and a trigger of premature senescence at least in human cells.