We used fetal bovine serum (FBS) and different growth factors to investigate their potential for inducing chondrogenic differentiation of synovium-derived stromal cells. Human synovium was harvested from patients suffering from osteoarthritis and expanded in monolayer. To evaluate the effect of serum and growth factors on chondrogenic differentiation, 10 ng/mL of transforming growth factor-beta1 (TGF-beta1), 100 ng/mL of bone morphogenic protein-2 (BMP-2), 100 ng/mL of insulin-like growth factor-1, 20 ng/mL of basic fibroblast growth factor (bFGF), and 10% FBS were added to the chemically defined chondrogenic medium singly or in combination during pellet culture for 21 days. The cell size and weight, glycosaminoglycan content, histology, and cartilage matrix-associated genes expression were analyzed. TGF-beta1 alone and TGF-beta1 + BMP-2 induced chondrogenic differentiation of synovium-derived stromal cells and synthesized cartilage-like matrix confirmed by histological analysis and immunohistochemistry. FBS, BMP-2, insulin-like growth factor-1, and bFGF as a single factor or other combinations except for TGF-beta1 + BMP-2 hardly induced chondrogenesis. Chondrogenic differentiation appeared to be inhibited when bFGF or the serum was added to the chondrogenic medium during pellet culture. The results of this study demonstrate the negative or positive role of serum and growth factors on chondrogenic differentiation of synovium-derived stromal cells.