Human mesenchymal stem cells (hMSCs), such as human adipose-derived stem cells (hADSCs), present heterogeneous characteristics, including varying differentiation abilities and genotypes. hADSCs isolated under different conditions exhibit differences in stemness. We isolated hADSCs from human fat tissues via culture on different cell culture biomaterials including tissue culture polystyrene (TCPS) dishes and extracellular matrix protein (ECM)-coated dishes in medium supplemented with 5% or 10% serum-converted human platelet lysate (hPL) or 10% fetal bovine serum (FBS) as a control. Currently, it is not clear whether xeno-free hPL in the cell culture medium promotes the ability of hMSCs such as hADSCs to differentiate into several cell lineages compared to the xenomaterial FBS. We investigated whether a synchronized effect of ECM (Matrigel, fibronectin, and recombinant vitronectin) coatings on TCPS dishes for efficient hADSC differentiation could be observed when hADSCs were cultured in hPL medium. We found that Matrigel-coated dishes promoted hADSC differentiation into osteoblasts and suppressed differentiation into chondrocytes in 10% hPL medium. Recombinant vitronectin- A nd fibronectin-coated dishes greatly promoted hADSC differentiation into osteoblasts and chondrocytes in 5% and 10% hPL media. hPL promoted hADSC differentiation into osteoblasts and chondrocytes compared to FBS on the fibronectin-coated surface and recombinant vitronectin-coated surface.