Articular cartilage regeneration remains challenging due to limited intrinsic repair capacity and the tendency of current human mesenchymal stem cell (hMSC)-based strategies to form fibrocartilage or undergo hypertrophy. Here, we present a defined biomaterial platform in which maltosebinding protein–fibroblast growth factor 2 (MBP-FGF2) is surface immobilized to create a bioinstructive interface that directs in vitro hMSC chondrogenesis without exogenous TGF-β1. The immobilized FGF2 interface promotes spontaneous, scaffold-free spheroid assembly via FGFR1–heparan sulfate proteoglycan–mediated adhesion, providing localized and sustained signaling. Transcriptomic and molecular analyses revealed early activation of FGF- and TGF-βrelated pathways, with upregulation of SOX9, COL2A1, and aggrecan and suppression of COL1A1 and COL10A1. These effects were consistently observed in hMSCs derived from both human embryonic stem cells and human tonsil tissue. Upon subcutaneous implantation into immunodeficient mice, pre differentiated spheroids generated ectopic cartilage tissue rich in type II collagen and aggrecan, even in the absence of TGF-β1. Taken together, these findings establish FGF2-immobilized biointerfaces as bioinstructive matrices that enable exogenous TGF-βindependent chondrogenesis and ectopic cartilage tissue formation, providing a defined and reproducible route for generating chondrogenic constructs for regenerative medicine.