Purpose Chondrocytes lose their ability to produce cartilaginous matrix during multiplication in culture through repeated passages, resulting in inferior tissue phenotype. To overcome the limited amount of primary chondrocytes, we aimed to determine the optimal culture condition for in vitro/in vivo cartilage regeneration using human adipose-derived mesenchymal stem cells (AMSCs). Methods To evaluate the effects exerted by the chondrocytic culture condition on AMSC, we utilized chondrocyte conditioned medium (CM) and/or co-culture methods to prime and differentiate AMSCs. We evaluated ultimate in vivo engineered cartilage with primed AMSCs with that of chondrocytes. To examine the link between conditioned factors and proliferation/differentiation, cell cycle progression of AMSCs were examined using 5-ethynyl-2'-deoxyuridine (EdU), and gene expression was monitored. Results We report that AMSCs can be stimulated to become chondrogenic cells when expanded with chondrocyte CM. Polymeric scaffolds co-seeded with CM-expanded AMSCs and primary chondrocytes resulted in in vivo cartilaginous tissues with similar biochemical content to constructs seeded with chondrocytes alone. Conclusion These results indicate that chondrocyte CM consists of suitable morphogenetic factors that induce the chondrogenic priming of AMSCs for cartilage tissue engineering.