The methods described herein allow for the isolation and expansion of fibroblastic-like ovine Wharton's jelly-derived mesenchymal stromal cells (oWJ-MSC) that, similarly to their human counterparts, adhere to standard plastic surfaces in culture; show a mesenchymal profile for specific surface antigens (i.e., positive for CD44 and CD166); and lack expression of endothelial (CD31) and hematopoietic (CD45) markers as well as major histocompatibility complex (MHC) class-II. Homogeneous cell cultures result from a two-phase bioprocess design that starts with the isolation of mesenchymal stromal cells (MSC) from the Wharton's jelly of ovine umbilical cords up to a first step of cryopreservation. The second phase allows for further expansion of ovine WJ-MSC up to sufficient numbers for further studies. Overall, this methodology encompasses a 2-week bioprocess design that encompasses two cell culture passages ensuring sufficient cells for the generation of a Master Cell Bank. Further thawing and scale expansion results in large quantities of oWJ-MSC that can be readily used in proof of efficacy and safety studies in the preclinical development stage of the development of cell-based medicines. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Isolation and expansion of ovine mesenchymal stromal cells from Wharton's jelly of the umbilical cord Basic Protocol 2: Characterization of ovine mesenchymal stromal cells Basic Protocol 3: Growth profile determination of ovine mesenchymal stromal cells from Wharton's jelly.