Adipose tissue as a stem cell source is ubiquitously available and has several advantages compared to other sources, for example it is easily accessible in large quantities with minimal invasive harvesting procedure, and isolation of adipose-derived mesenchymal stem cells (MSCs) yields a high amount of stem cells, essential for stem cell-based therapies and tissue engineering. We have explored the effect of donor age, and the anatomical origin of the adipose tissue on several aspects of MSCs in dogs, such as cell yield, proliferative ability, multi-differentiation potential, colony-forming capacity, stemness marker expression. We also assessed the effect of cell passaging on the MSCs stemness. We found that the anatomical origin of the adipose tissue and the age of donors have effects only on the proliferative capacity of the MSCs. Moreover, cells show a progressive loss of the stemness characteristics with passages. Cell therapies need a suitable number of cells to use in clinical applications. Characterization of MSCs at different passages, allowed us to demonstrate that, under our culture conditions, the best quantitative and qualitative characteristics are obtained at early passages. Adult MSCs are of particular interest for the therapeutic approach to musculoskeletal diseases, and the dog provides an excellent preclinical model for the development of new approaches in regenerative medicine that might be applied to humans.