INTRODUCTION: Mesenchymal stem cells (MSCs) possess immunomodulatory potential and are used for cell therapy in both human and veterinary medicine. However, donor-derived MSCs have limited proliferative activities and variations, which restrict their clinical applicability. In contrast, induced pluripotent stem cells (iPSCs) can self-renew indefinitely and differentiate into the three germ layers. By exploiting these characteristics, iPSCs can differentiate into mesenchymal stem cells (iMSCs) and potentially overcome the limitations of donor-derived MSCs. In humans, the characteristics of iMSCs have been reported to vary depending on the differentiation strategy and cell origin of iPSCs. However, no studies have investigated the differentiation strategies and cell origins of canine iPSCs (ciPSCs) in relation to iMSC generation. METHODS: Canine embryonic fibroblast-derived iPSCs (CEF-iPSCs) were differentiated into iMSCs via the mesoderm or ectoderm, and their proliferative ability and the expression levels of CD34, CD44, CD45, and CD90 were assessed. We then applied the iMSC induction method via the ectoderm to other ciPSC lines, including canine dermal fibroblast-derived iPSCs (CDF-iPSCs), canine peripheral mononuclear cell-derived iPSCs (cPBMC-iPSCs), and canine urine-derived cell-derived iPSCs (cUC-iPSCs). We assessed their proliferation, marker expression, and ability to differentiate into tri-lineages and performed comparative analyses. RESULTS: IMSCs derived from CEF-iPSCs via the ectodermal lineage showed higher proliferative ability and expressed MSC markers at a higher rate than iMSCs generated via mesodermal induction. Notably, with the exception of CDF-iPSCs, iMSCs were successfully generated from other ciPSC lines via ectodermal lineages. These iMSCs exhibited proliferative activities over passage 10, expressed MSC markers, and demonstrated the ability to differentiate into tri-lineages. iMSCs derived from cUC-iPSCs exhibited the highest expression of CD90 compared to other iMSCs. CONCLUSIONS: Highly proliferative iMSCs expressing a high rate of MSC markers can be obtained from cUC-iPSCs via ectodermal induction. Our study demonstrated that the differentiation strategy and cell origin of ciPSCs play crucial roles in the generation of iMSCs.