Stem cell therapy using mesenchymal stem/stromal cells (MSCs) represents a novel approach to treating severe diseases, including osteoarthritis. However, the therapeutic benefit of MSCs is highly dependent on their differentiation state, which can be regulated by many factors. Herein, three-dimensional (3D) magnetic scaffolds were successfully fabricated by incorporating magnetic nanoparticles (MNPs) into electrospun gelatin nanofibers. When positioned near a rotating magnet (= 0.5 Hz), the magnetic scaffolds with the embedded MSCs were driven upward/downward in the culture container, which induced mechanical stimulation to MSCs due to spatial confinement and fluid flow. The extracellular matrix-mimicking scaffold and the alternating magnetic field significantly enhanced chondrogenesis instead of osteogenesis. Furthermore, the fiber topography could be tuned with different compositions of the coating layer on MNPs, and the topography had a significant impact on MSC differentiation. Selective up-regulation of chondrogenesis-related genes (and) was found for the magnetic scaffolds with citric acid-coated MNPs (CAG). In contrast, osteogenesis-related genes (and) were selectively and significantly up-regulated for the magnetic scaffolds with polyvinylpyrrolidone-coated MNPs. Prior to implantation, chondrogenic preconditioning of MSCs within the CAG scaffolds under a dynamic magnetic field resulted in superior osteochondral repair. Hence, the magnetic scaffolds together with an in-house rotating magnet device could be a novel platform to initiate multiple stimuli on stem cell differentiation for effective repair of osteochondral defects.