The possibility to isolate synovial fluid-derived mesenchymal stem cells (SFMSCs) from patients with degenerative arthropathy has been an interest since synovial fluid (SF) from osteoarthritis (OA) patients offered a unique stem-cell resource for therapeutic applications. In this study, we successfully isolated, cytogenetically and molecularly characterized, and followed the differentiation potency of human mesenchymal stem cells (MSCs) from SF. The morphology of proliferating SFMSCs showed fibroblast-like morphology, and both the population doubling time (DT) and viability of MSCs from bone marrow, adipose, and SF did not differ. The immunophenotype of SFMSCs was confirmed by the positive expression of CD44, CD73, CD90, CD105, and CD106 by flow cytometry and immunocytochemistry, and the expression of the hematopoietic markers, CD34 and CD45, was not found. In all MSCs from three different origins, we measured the mRNA expression of developmentally important transcript factors such as KLF4, c-Myc, Sox2, and OCT4. SFMSCs from OA patients showed normal chromosomal number, structure, and telomerase activity. SFMSCs showed multipotent capacity, and was differentiated into neurocyte, adipocyte, osteocyte, and chondrocyte in vitro, as demonstrated by specific stains and expression of molecular markers. In addition, SFMSCs also have the capacity to secrete immunomodulating factors (IL-4, IL-10, IL-13, and transforming growth factor-${eta}$ (TGF-${eta}$)) involved in the therapy of rheumatoid arthritis (RA). These results demonstrate that SFMSCs from OA-patients might provide therapeutic options for RA and OA.