Abstract: The present study has been undertaken for isolation, characterization and differentiation of Buffalo Adipose Derived Mesenchymal Stem Cells (bADMSCs). Cocktail enzymatic digestion method was used for isolation of bADMSCs which yielded optimum number of cells than other methods. Subcutaneous adipose tissue yielded comparatively higher number of Stem Cells (SC) than from omentum and bone regions. The growth and proliferation of SC were rapid when Mesencult medium was used, in comparison to Dulbecco’s Modified Eagles Medium (DMEM). Expression of oct 4 gene by reverse transcriptase-polymerase chain reaction (RT-PCR) indicated the stemness of isolated bADMSCs. Magnetic cell sorting and immunostaining revealed that the isolated ADMSCs were cluster of differentiation 44 (CD44) positive. Buffalo ADMSCs were also induced to differentiate into adipogenic, osteogenic and chondrogenic lineages. Expression of specific marker genes-Peroxisome Proliferator Activated Receptor Gamma (PPAR γ) in adipogenic, Osteopontin in osteogenic and Collagen II in chondrogenic differentiated lineages were confirmed in buffalo Adipose Derived Stem Cells (ADSCs) by RT-PCR. The study clearly proves that Buffalo white adipose tissue is a source of multipotent SC and can be induced to differentiate into adipogenic, osteogenic and chondrogenic lineages. Further, this is the first study to report such finding in buffalo.