The dog is an underrepresented large animal translational model for orthopedic cell-based tissue engineering. While chondrogenic differentiation of canine multipotent stromal cells (cMSCs) has been reported using the classic micromass technique, cMSCs respond inconsistently to this method. The objectives of this study were to develop a three-dimensional (3D), serum-free, Collagen Type I system to facilitate cMSC chondrogenesis and, once established, to determine the effect of chondrogenic growth factors on cMSC chondrogenesis. Canine MSCs were polymerized in 100 μL Collagen Type I gels (5 mg/mL) at 1 x 106 cells/construct. Constructs were assessed using morphometry, live/dead staining, and histology in 10 various chondrogenic media. Four media were selected for additional in-depth analyses via lactate dehydrogenase release, total glycosaminoglycan content, qPCR (COL1A1, COL2A, SOX9, ACAN, BGLAP and SP7), immunofluorescence, and TUNEL staining. In the presence of dexamethasone and transforming growth factor-β3 (TGF-β3), both bone morphogenic protein-2 (BMP-2) and basic fibroblast growth factor (bFGF) generated larger chondrogenic constructs, although BMP-2 was required to achieve histologic characteristics of chondrocytes. Chondrogenic medium containing dexamethasone, TGF-β3, BMP-2 and bFGF led to a significant decrease in lactate dehydrogenase release at day 3 and glycosaminoglycan content was significantly increased in these constructs at day 3, 10, and 21. Both osteogenic and chondrogenic transcripts were induced in response to dexamethasone, TGF-β3, BMP-2 and bFGF. Collagen Type II and X were detected in all groups via immunofluorescence. Finally, TUNEL staining was positive in constructs lacking BMP-2 or bFGF. In conclusion, the 3D, serum-free, Collagen Type-I assay described herein proved useful in assessing cMSC differentiation and will serve as a productive system to characterize cMSCs or to fabricate tissue engineering constructs for clinical use.