Chondrogenic differentiation of mesenchymal stem cells (MSCs) within a three-dimensional (3D) environment can be guided to form cartilage-like tissue in vitro to generate cartilage grafts for implantation. 3D bioprinted, MSC-populated cartilage grafts have the potential to replace autologous cartilage in reconstructive airway surgery. Here, bone marrow-derived ferret MSCs (fMSCs) capable of directed musculoskeletal differentiation were generated for the first time. A multi-material, 3D bioprinted fMSC-laden scaffold was then engineered that was capable of in vitro cartilage regeneration, as evidenced by glycosaminoglycan (GAG) production and collagen II immunohistochemical staining. In vivo implantation of these 3D bioprinted scaffolds in a ferret model of laryngotracheal reconstruction (LTR) demonstrated healing of the defect site, epithelial mucosalization of the inner lumen, and expansion of the airway volume. While the implanted scaffold allowed for reconstruction of the created airway defect, minimal chondrocytes were identified at the implant site. Nevertheless, we have established the ferret as biomedical research model for airway reconstruction and, although further evaluation is warranted, the generation of fMSCs provides an opportunity for realizing the potential for 3D bioprinted regenerative stem cell platforms in the ferret.