ObjectiveAdipose tissue–derived stem cells (ASCs) are a promising source of cells for articular cartilage regeneration. However, ASCs isolated from different adipose tissue depots have heterogeneous cell characterizations and differentiation potential when cultured in 3-dimensional (3D) niches.DesignWe compared the chondrogenicity of ASCs isolated from infrapatellar fat pads (IPFPs) and subcutaneous fat pads (SCFPs) in 3D gelatin-based biomimetic matrix.ResultsThe IPFP-ASC-differentiated chondrocytes had higher <i>ACAN</i>, <i>COL2A1</i>, <i>COL10</i>, <i>SOX6</i>, <i>SOX9</i>, <i>ChM-1</i>, and <i>MIA-3</i> mRNA levels and lower <i>COL1A1</i> and <i>VEGF</i> levels than the SCFP-ASCs in 3D matrix. The difference in mRNA profile may have contributed to activation of the Akt, p38, RhoA, and JNK signaling pathways in the IPFP-ASCs. The chondrocytes differentiated from IPFP-ASCs had pronounced glycosaminoglycan and collagen type II production and a high chondroitin-6-sulfate/chondroitin-4-sulfate ratio with less polymerization of β-actin filaments. In an <i>ex vivo</i> mice model, magnetic resonance imaging revealed a shorter T2 relaxation time, indicating that more abundant extracellular matrix was secreted in the IPFP-ASC–matrix group. Histological examinations revealed that the IPFP-ASC matrix had higher chondrogenic efficacy of new cartilaginous tissue generation as evident in collagen type II and S-100 staining. Conclusion. ASCs isolated from IPFPs may be better candidates for cartilage regeneration, highlighting the translational potential of cartilage tissue engineering using the IPFP-ASC matrix technique.