The objective was to improve proliferation and chondrogenic potential of adipose stem cells (ASCs) by expansion on extracellular matrix (ECM) deposited by either ASCs or synovium-derived stem cells (SDSCs). ASCs isolated from porcine infrapatellar fat pad were separately expanded on conventional plastic flasks, ASC-deposited ECM and SDSC-deposited ECM. ASCs were centrifuged to form pellets and cultured in a serum-free chondrogenic medium with either TGFβ3 or TGFβ3 combined with BMP-6. Cell number yielded on ECM expansion did not show a significant difference in deposition between ASCs and SDSCs but was 6–10 times that grown on non-coated flasks. ECM-expanded ASCs exhibited a lower level of intracellular reactive oxygen species (ROS) compared to those grown on non-coated flasks. Typical chondrogenic markers, including type II collagen and glycosaminoglycans (GAGs), were intensively distributed in the pellets from ECM-expanded ASCs instead of those from flask-grown cells. ASCs expanded on ECM, either from ASCs or SDSCs, exhibited a similar chondrogenic index (GAG:DNA), which was significantly higher than that from ASCs grown on non-coated flasks. The combination of TGFβ3 and BMP-6 increased 36% more in ASC chondrogenic index than the treatment with TGFβ3 alone. Interestingly, ECM pretreatment also decreased expanded ASC hypertrophic marker genes. ECM deposited by either ASCs or SDSCs did not exhibit enhanced adipogenic differentiation of ASCs. Our study indicates that the sequential application of ECM for cell expansion and combined TGFβ3 with BMP-6 for chondrogenic differentiation may be a promising approach for ASC-based cartilage tissue engineering and regeneration. Copyright © 2011 John Wiley & Sons, Ltd.