OBJECTIVE Tracheal reconstruction using a stem cell-based engineered trachea has recently shown promise. Our goal is to achieve a single-stage stem cell-based tracheal replacement. STUDY DESIGN Prospective feasibility study. SETTING Wake Forest Institute of Regenerative Medicine. SUBJECTS AND METHODS Five healthy male beagles were implanted with a 2.5-cm segment of decellularized trachea. A sixth animal, planned for the control arm, died of anesthetic complications prior to tracheal implantation. The remaining 5 beagles were divided into 2 study arms: 4 had adipose-derived stem cells coating the lumen of the donor trachea, and a control animal had the trachea implanted cell free. The donor tracheas were obtained from previously sacrificed size-matched canines and decellularized. The adipose tissue was harvested from a recipient animal and the trachea prepared, seeded, and then implanted, all in one operation. Adipose stem cells were labeled fluorescently. RESULTS Five of 6 planned surgical procedures were completed successfully. All required sacrifice for airway distress at approximately 1 week postoperatively. All tracheal grafts were found to be malacic and compromised. CONCLUSION In a canine model using a decellularized tracheal scaffold and adipose stem cells, the postoperative inflammatory response and evidence of rejection was minimal. However, all scaffolds exhibited breakdown, compromising the animals' airways, necessitating euthanasia earlier than planned. For future study, a similar animal model using a single-stage approach with a more robust scaffold may allow for greater survival and stem cell differentiation.