The study characterized mesenchymal stem cells (MSCs) derived from human synovial membrane (SM) and demonstrated their multilineage differentiation potential. SM-derived cells were expanded in monolayer with limited senescence, and their multilineage potential was investigated through chondrogenesis, osteogenesis, myogenesis, and adipogenesis assays. The cells showed extensive expandability, with no significant telomerase activity detected, but maintained a linear growth curve up to 30 population doublings. Senescence was minimal, with only a few cells showing detectable senescence-associated β-galactosidase (SA-β-gal) activity at late passages. The molecular profile of SM-derived cells remained stable during expansion, with rapid disappearance of hematopoietic and endothelial markers. Chondrogenic, osteogenic, myogenic, and adipogenic differentiation was induced in all tested donor samples, regardless of donor age or cell passage number. Individual SM-derived cell clones also exhibited multilineage potential, with only one clone showing myogenic differentiation. The findings suggest that SM-derived MSCs have significant therapeutic potential for regenerative medicine, particularly in joint surface defect repair, as they are easily accessible and can be stored for long-term use.The study characterized mesenchymal stem cells (MSCs) derived from human synovial membrane (SM) and demonstrated their multilineage differentiation potential. SM-derived cells were expanded in monolayer with limited senescence, and their multilineage potential was investigated through chondrogenesis, osteogenesis, myogenesis, and adipogenesis assays. The cells showed extensive expandability, with no significant telomerase activity detected, but maintained a linear growth curve up to 30 population doublings. Senescence was minimal, with only a few cells showing detectable senescence-associated β-galactosidase (SA-β-gal) activity at late passages. The molecular profile of SM-derived cells remained stable during expansion, with rapid disappearance of hematopoietic and endothelial markers. Chondrogenic, osteogenic, myogenic, and adipogenic differentiation was induced in all tested donor samples, regardless of donor age or cell passage number. Individual SM-derived cell clones also exhibited multilineage potential, with only one clone showing myogenic differentiation. The findings suggest that SM-derived MSCs have significant therapeutic potential for regenerative medicine, particularly in joint surface defect repair, as they are easily accessible and can be stored for long-term use.