Trend of regenerative therapy for diabetes in human and veterinary practice has conceptually been proven according to Edmonton protocol and animal models. Establishing an alternative insulin-producing cell (IPC) resource is a challenge task for further clinical application. In this study, IPC generation from two practical canine mesenchymal stem cells(cMSCs), canine bone marrow-derived MSCs (cBM-MSCs) and canine adipose-derived MSCs(cAD-MSCs), was of interest. The results illustrated that cBM-MSCs and cAD-MSCs contained distinct pancreatic differentiation potential and required the tailor-made induction protocols. Effective generation of cBM-MSC-derived IPCs needed an integration of genetic and microenvironment manipulation using hanging-drop culture of PDX-1-transfected cBM-MSCs under three-step pancreatic induction protocol. However, this protocol was resource- and time-consumed. Another study on cAD-MSC-derived IPC generation found that IPC colonies could be obtained by low attachment culture under three-step induction protocol. Further Notch signaling inhibition during pancreatic endoderm/progenitor induction yielded IPC colonies with trend of glucose-responsive C-peptide secretion. Thus, this study showed that IPCs could be obtained from cBM-MSCs and cAD-MSCs by different induction techniques, and further signaling manipulation study should be conducted to maximize the protocol efficiency.