This article aims to explore whether Wharton’s jelly (WJ) derived mesenchymal stem cells (MSCs) (WJ-MSCs) decellularized extracellular matrix (dECM) can rejuvenate MSCs during in vitro expansion. Passage 10 synovium-derived mesenchymal stem cells (SDSCs) and WJ-MSCs were expanded on plastic flasks (PL) or dECMs derived from SDSCs and WJ-MSCs. Flow cytometry was applied to evaluate surface phenotypes and proliferation capacity. Early (7 days) and late (21 days) chondrogenic potentials were assessed using histology, immunohistochemistry, and real-time polymerase chain reaction (PCR). Western blot analysis was applied to evaluate the potential involvement of MAPK and Wnts signals during the proliferation and chondrogenic processes. Cells were further evaluated for their osteogenic potential using alkaline phosphatase staining and RT-PCR and adipogenic potential using oil red O staining and RT-PCR. Compared to PL expanded cells, dECMs yielded expanded cells with better proliferation capacity as well as decreased percentage of HLA-DR positive SDSCs. Meanwhile, a decrease in CD105 median fluorescence intensity of WJ-MSCs groups were observed compared to the corresponding SDSCs groups. Moreover, both SDSCs and WJ-MSCs acquired better chondrogenic potential after dECM treatment, as evidenced by increased pellet sizes and increased expression of chondrogenic marker genes. WJ-MSCs dECM was inferior to SDSCs dECM in enhancing early stage chondrogenic differentiation, which was compensated during late stage chondrogenesis, despite causing an increased type X collagen accumulation. p-JNK and p-38 were implicated in the expansion and late chondrogenic differentiation stages, respectively. However, dECM preconditioning did not enhance either osteogenic or adipogenic potential of SDSCs and WJ-MSCs. WJ-MSCs dECM is superior to SDSCs dECM on enhancing proliferation, lowering immunogenicity and promoting late stage chondrogenesis.