Background <p>Mesenchymal stromal cells (MSCs) hold significant potential for various applications in regenerative medicine and tissue engineering. Initially considered as a single cell type with defined characteristics, MSCs are now known as a heterogeneous cell population with remarkable differences in their properties. No consensus exists on how donor age affects MSC characteristics, like proliferation. Additionally, differences in differentiation capacities and immunophenotype could arise when MSCs are isolated from different animals breeds, which is relevant for experimental and preclinical studies of MSC-based treatments.</p> Methods <p>In this study, we isolated bovine adipose tissue-derived MSCs from three age categories, i.e. fetal, calf, and adult, and of two different breeds, i.e. Holstein Friesian (HF) and Belgian Blue (BB). MSC characterization included tri-lineage differentiation, proliferation and senescence assays, and immunophenotyping using multi-color flow cytometry.</p> Results <p>Especially fetal and calf HF-MSCs showed a high proliferation capacity, where 4 and 6 out of 7 donors, respectively, could surpass 30 population doublings. Adipogenic differentiation potential was higher for fetal and adult HF-MSCs. Furthermore, breed, but not age, affected their osteogenic differentiation potential, with BB-MSCs performing better. Evaluation of cell surface marker expression revealed a breed effect, as calf HF-MSCs showed a higher percentage of Cluster of Differentiation (CD)34⁺ cells compared to calf BB-MSCs, which was correlated with both osteogenic differentiation and proliferation potential.</p> Conclusions <p>Our findings clearly show the impact of donor characteristics such as age and breed on MSC proliferation, immunophenotype, and differentiation potential, illustrating the importance of selecting the appropriate MSC donor for MSC-based treatments when allogeneic MSCs are considered.</p>