Bone marrow-derived mesenchymal stromal stem cells (BMSCs) are a promising cell source for treating articular cartilage defects (Bornes et al., 2014). BMSCs can be seeded within porous biomaterial scaffolds that support three-dimensional cell organization, chondrogenic differentiation and extracellular matrix deposition for the creation of engineered cartilage. This protocol describes our defined methods for isolation and expansion of human and ovine BMSCs, seeding of BMSCs within porous scaffolds and in vitro chondrogenic differentiation (Adesida et al., 2012; Bornes et al., 2015).