We previously found that the combination of an autologous bone mesenchymal stem cell-derived extracellular matrix (aBMSC-dECM) scaffold with bone marrow stimulation could enhance hyaline cartilage regeneration. We suspected that chondrogenic differentiation could be induced by the aBMSC-dECM scaffold. This study aimed to investigate whether aBMSC-dECM scaffolds could promote chondrogenic differentiation without exogenous growth factors. BMSCs were seeded on aBMSC-dECM scaffolds and cultured in vitro with or without transforming growth factor-β3 (E(+) or E(-) group). Atelocollagen scaffolds were used as controls (C(+) or C(-) group). The chondrogenic differentiation was evaluated by histological, biochemical, and real-time polymerase chain reaction assays. After 3 weeks, cartilage-like tissue with a homogeneous structure, a high cartilaginous matrix content (proteoglycan and type II collagen), and high expression levels of cartilage-associated genes (COL2A1, ACAN, and SOX9) were observed in the E(+), E(-), and C(+) groups. In addition, BMSCs in each scaffold (E group or C group) were preconditioned with chondrogenic media in vitro for 1 week, and then implanted in the backs of nude mice for 3 weeks. Three weeks later, cartilage matrix formation (proteoglycan and type II collagen) was achieved only in the E group, confirmed by safranin O staining and immunohistochemical staining for type II collagen. Taken together, these results indicate that aBMSC-dECM scaffolds could induce chondrogenic differentiation. Thus, they could be successful candidate scaffolds for cartilage tissue engineering.