During the last decade, many cartilage tissue engineering strategies have been developed, being the stem cell-based approach one of the most promising. Transforming Growth Factor-β3 (TGF-β3) and Insulin-like Growth Factor-I (IGF-I) are key proteins involved in the regulation of chondrogenic differentiation. Therefore, these two growth factors (GFs) were immobilized at the surface of a single electrospun nanofibrous mesh (NFM) aiming to differentiate human Bone Marrow-derived Mesenchymal Stem Cells (hBM-MSCs). The immobilization of defined antibodies (i.e. anti-TGF-β3 and anti-IGF-I) allows the selective retrieval of the abovementioned GFs from human platelet lysates (PL). Biochemical assays, involving hBM-MSCs cultured on biofunctional nanofibrous substrates under basal culture medium during 28 days, confirm the biological activity of bound TGF-β3 and IGF-I. Specifically, the typical spherical morphology of chondrocytes and the immunolocalization of collagen type II confirmed the formation of a cartilaginous ECM. Therefore, the proposed biofunctional nanofibrous substrate is able to promote chondrogenesis.