Recent endeavors to use stem cells as trophic factor production sources have the potential to translate into viable therapies for damaged or diseased musculoskeletal tissues. Adipose stem cells (ASCs) can be differentiated into chondrocytes using the chondrogenic medium (CM), but it is unknown if this approach can optimize ASC growth factor secretion for cartilage regeneration by increasing the chondrogenic factor production, while decreasing angiogenic and hypertrophic factor production. The objective of this study was to determine the effects the CM and its components have on growth factor production from ASCs to promote cartilage regeneration. ASCs isolated from male Sprague-Dawley rats and cultured in monolayer or alginate microbeads were treated with either the growth medium (GM) or the CM for 5 days. In subsequent studies, ASC monolayers were treated with either the GM supplemented with different combinations of 50 μg/mL ascorbic acid-2-phosphate (AA2P), 100 nM dexamethasone (Dex), 10 ng/mL transforming growth factor (TGF)-β1, and 100 ng/mL bone morphogenetic protein (BMP)-6 or with the CM excluding different combinations of AA2P, Dex, TGF-β1, and BMP-6. mRNA levels and growth factor production were quantified at 8 and 24 h after the last media change, respectively. The CM increased chondrogenic factor secretion (TGF-β2, TGF-β3, and insulin-like growth factor [IGF]-I) and decreased angiogenic factor production (the vascular endothelial growth factor [VEGF]-A, the fibroblast growth factor [FGF]-2). Microencapsulation in the GM increased production of the chondrogenic (IGF-I, TGF-β2) and angiogenic (VEGF-A) factors. AA2P increased secretion of chondrogenic factors (IGF-I, TGF-β2), and decreased angiogenic factor (VEGF-A) secretion, in addition to decreasing mRNA levels for factors associated with chondrocyte hypertrophy (FGF-18). Dex increased mRNA levels for hypertrophic factors (BMP-2, FGF-18) and decreased angiogenic factor secretion (VEGF-A). TGF-β1 increased angiogenic factor production (FGF-2, VEGF-A) and decreased chondrogenic factor mRNA levels (IGF-I, PTHrP). BMP-6 increased hypertrophic mRNA levels (FGF-18) and chondrogenic factor production (TGF-β2). When ASC microbeads preconditioned with the CM were implanted in a focal cartilage defect and immobilized within an RGD-conjugated hydrogel, tissue infiltration from the edges of the defect and perichondrium was observed. These results show that differentiation media components have distinct effects on ASC's production of angiogenic, chondrogenic, and hypertrophic factors and that AA2P may be the most beneficial CM component for preconditioning ASCs to stimulate cartilage regeneration.