Articular cartilage is a tissue that shows no capacity for effective spontaneous regeneration. Tissue engineering, as a new important field of regenerative medicine, emerges as potential effective solution for treatment of articular cartilage injuries and defects. The aim of this study was to optimize conditions for 3D in vitro chondrogenesis. In the experiment, three different cell culture types were used: chondrocytes, human bone marrow mesenchymal stem cells (hBMSCs) and combination of chondrocytes and hBMSCs in 2:1 ratio. Cells were grown in a chondrogenic medium, incorporated in a peptide hydrogel RADA, in either normoxic (20% O2) or hypoxic (5% O2) conditions. Dexamethasone, one of the chondrogenic medium components, was added in a differentiation medium or it was incorporated with cells within a peptide hydrogel. After 19 days, expression levels of two cartilage marker genes, SOX9 and ACAN, were evaluated by quantitative PCR. Chondrocytes showed the highest and hBMSCs the lowest chondrogenic potential among used cell types. Hypoxia enhanced chondrogenesis significantly only in the case of chondrocytes, while dexamethasone source did not considerably influence on expression levels of SOX and ACAN genes.