Perlecan (HSPG2), a key component of basement membrane proteins, plays a crucial role in tissue development and regeneration. However, its direct impact on mesenchymal tissue differentiation, mediated through both genetic modification (gain- and loss-of-function mutations) and epigenetic changes (matrix microenvironment alterations), remains underexplored. In this study, we utilized CRISPR/Cas9 to achieve knockout (KO) and overexpression (OE) of HSPG2 in human fetal nucleus pulposus stem/progenitor cells (NPSCs) and adult infrapatellar fat pad-derived stem cells (IPFSCs) to investigate perlecan's influence on mesenchymal differentiation. We also assessed the effects of decellularized extracellular matrix (dECM) derived from fetal NPSCs with modified HSPG2 expression on the proliferation and differentiation of adult NPSCs. Our findings demonstrate that HSPG2-KO enhanced chondrogenic differentiation, while HSPG2-OE suppressed adipogenic differentiation in both fetal NPSCs and adult IPFSCs. Notably, dECM from HSPG2-OE fetal NPSCs significantly promoted chondrogenic differentiation in adult NPSCs, suggesting potential applications for perlecan in developing advanced biomaterials for cartilage regeneration.