Articular cartilage is one of the most important weight-bearing components in human body, thus the chondrogenesis of stem cells is reactive to many intracellular and extracellular mechanical signals. As a unique physical cue, matrix stiffness plays an integral role in commitment of stem cell fate. However, when examining the downstream effects of matrix stiffness, most studies used different soluble factors to assist physical inducing process, which may mask the chondrogenic effects of matrix stiffness. Here we fabricated polyacrylamide (PAAm) hydrogels with gradient stiffness to unravel the role of matrix stiffness in chondrogenic process of mesenchymal stem cells (MSCs), with or without TGF-β3 as induction factor. The results showed that with micromass culture mimicking relatively high cell density in vivo, the chondrogenic differentiation of MSCs can be promoted by soft substrates (about 0.5 kPa) independently with assembled cytoskeleton. Further analysis indicated that addition of TGF-β3 generally increased expression level of cartilage-related markers and masked the stiffness-derived expression pattern of hypertrophic markers. These results demonstrate how mechanical cues experienced in developmental context regulate commitment of stem cell fate and have significant impact on the design of tissue regeneration materials.