The data obtained in this study indicate that mesenchymal stromal cells derived from amniotic fluid (AF-MSCs) may be a promising source for articular cartilage regeneration. The cells were differentiated into chondrocytes in a three-dimensional culture system, forming cellular spheroids using a 3D-printed micromold composed of non-adhesive hydrogel. The spheroids demonstrated good uniformity, cell viability, and extracellular matrix production capability, with the synthesis of type II collagen and aggrecan, characteristic of cartilage.