The precise spatial and temporal presentation of growth factors is critical for cartilage development, during which tightly controlled patterns of signals direct cell behavior and differentiation. Recently, chondrogenic culture of human mesenchymal stem cells (hMSCs) has been improved through the addition of polymer microspheres capable of releasing growth factors directly to cells within cellular aggregates, eliminating the need for culture in transforming growth factor-β1 (TGF-β1)-containing medium. However, the influence of specific patterns of spatiotemporal growth factor presentation on chondrogenesis within microsphere-incorporated cell systems is unclear. In this study, we examined the effects of altering the chondrogenic microenvironment within hMSC aggregates through varying microsphere amount, growth factor concentration per microsphere, and polymer degradation time. Cartilage formation was evaluated in terms of DNA, glycosaminoglycan, and type II collagen in hMSCs from three donors. Chondrogenesis equivalent to or greater than that of aggregates cultured in medium containing TGF-β1 was achieved in some conditions, with varied differentiation based on the specific conditions of microsphere incorporation. A more spatially distributed delivery of TGF-β1 from a larger mass of fast-degrading microspheres improved differentiation by comparison with delivery from a smaller mass of microspheres with a higher TGF-β1 concentration per microsphere, although the total amount of growth factor per aggregate was the same. Results also indicated that the rate and degree of chondrogenesis varied on a donor-to-donor basis. Overall, this study elucidates the effects of varied conditions of TGF-β1-loaded microsphere incorporation on hMSC chondrogenesis, demonstrating that both spatiotemporal growth factor presentation and donor variability influence chondrogenic differentiation within microsphere-incorporated cellular constructs.