Joint injury results in cartilage lesions that are characterized by a poor repair response. Adult stem cells are immensely appealing for biological joint repair, such as cartilage tissue engineering and regeneration. However, adult stem cells gradually lose their stemness once they are removed from their in vivo niche for plating in plastic flasks. We utilized a tissue-specific stem cell, synovium-derived stem cell (SDSC), as a model to reconstruct an in vitro three-dimensional stem cell niche. After seeding on SDSC-derived extracellular matrix, the initially wide and flat SDSCs became thin and spindle shaped and were arranged in a three-dimensional configuration with typical stem cell phenotypes. A dramatic increase in cell number and a greatly enhanced chondrogenic capacity were observed, though surprisingly the extracellular matrix-treated SDSCs did not display concomitantly improved adipogenic or osteogenic potentials. Thus, we conclude that a tissue-specific stem cell can be used to prepare its own in vitro niche for stem cell proliferation while maintaining and enhancing its lineage-specific stemness. The ability to reconstitute the in vitro stem cell niche will greatly benefit SDSC-based therapy for cartilage defects.