Background　aims.　Multipotent　mesenchymal　stromal　cells　(MSCs)　are　promising　candidates　for　innovative　cell　therapeutic　applications.　Before　their　use,　however,　they　usually　need　to　be　expanded　in　vitro　with　serum-supplemented　media.　MSCs　can　undergo　replicative　senescence　during　in　vitro　expansion,　but　it　is　not　yet　clear　how　serum　supplements　influence　this　process.　Methods.　In　the　present　study,　we　compared　how　Media　supplemented　with　fetal　bovine　serum　(PBS)　or　calf　serum　(CS)　affected　morphology,　proliferation,　differentiation,　senescence　and　other　functional　characteristics　of　human　umbilical　cord-derived　MSCs　(UC-MSCs).　Results.　UC-MSCs　cultured　in　both　FBS-　and　CS-containing　media　were　able　to　differentiate　along　osteogenic　and　adipogenic　lineages　but　ultimately　reached　proliferation　arrest.　However,　senescence-associated　characteristics,　such　as　beta-galactosidase　activity,　reactive　oxygen　species　levels,　proliferation　rate　and　gene　expression,　demonstrate　that　UC-MSCs　grown　with　FBS　have　better　proliferation　potential　and　differentiation　capacity.　In　contrast,　UC-MSCs　grown　with　CS　have　a　higher　proportion　of　apoptotic　cells　and　senescent　characteristics.　Possible　mechanisms　for　the　observed　phenotypes　include　changes　in　gene　expression　(Bax　p16,　p21　and　p53)　and　cytokine　production　(interleukin-6　and　interleukin-8).　Conclusions.　This　study　demonstrates　that　FBS-supplemented　media　provides　a　better　microenvironment　for　the　expansion　of　UC-MSCs　in　vitro　than　CS-supplemented　media.　This　work　provides　insight　into　MSCs　generation　practices　for　use　in　basic　research　and　clinical　therapies.