In recent years, various clinical trials are exploring the efficacy of cell-based strategies to treat cartilage defects. Human adipose-derived stem/stromal cells (hASCs) have great potential for the regeneration of articular cartilage due to their ability to undergo differentiation into the chondrogenic lineage. In most cell-based therapies, the single dose requires 10 7 -10 9 cells, and then it is necessary to move from manual flask-based culture to bioreactor whose monitoring and control of biological variability is necessary. Furthermore, before clinical use, the success of hASC differentiation need to be thoroughly inspected. In this study, we sought to assess with complementary methods the chondrogenic potential of hASCs expanded in flask or in a hollow-fiber bioreactor (HFBR). Human ASCs, after phenotypic characterization were subjected to chondrogenic differentiation, the expression of cartilage-specific genes was analysed by quantitative real-time polymerase chain reaction (qPCR); optical microscopy as well as transmission electron microscopy (TEM) were also performed. The two culture expansion systems do not affect hASC immunophenotype; microscopy analysis confirmed the successful of hASC differentiation process. Gene expression analysis after differentiation displayed an upregulation of cartilage-specific genes more noticeable in hASCs expanded in the HFBR, suggesting that hASCs expanded in the HFBR can be efficiently differentiated into chondroblasts. Our study demonstrates that flask and HFBR expansion methods do not affect the subsequent differentiation process; additionally, our results highlight the importance of analyzing differentiation by different techniques and support the importance of TEM analysis that is able to give additional critical information to molecular biology results.