In this case series, a tissue-engineered bilayered live cell therapy (LCT) is examined as an alternative to free gingival grafts in subjects with oral mucosal defects. Four patients with five sites requiring mucogingival surgery because of limited vestibular depth and limited keratinized oral mucosa were selected for treatment. All included patients had limited graft availability or preferred to avoid autogenous soft tissue grafting. The oral mucosal defects were treated with a tissueengineered bilayered LCT, a circular disk of allogeneic live, human-derived, confluent, and bilayered keratinocyte and fibroblast cells. Sites were evaluated for wound healing, histologic appearance of the regenerated mucosa, and DNA persistence of the LCT. All sites healed uneventfully, with complete epithelialization by 14 days postoperative and no significant adverse events. The treated sites matched the surrounding tissues in both texture and color. Histologic samples revealed gingival tissue architecture characterized by a parakeratinized epithelium with a very sparse and diffuse chronic inflammatory response within the connective tissue. DNA persistence indicated that the pairs of samples (swab and biopsy) collected from each subject displayed the same identifier DNA, with no evidence of any other source. Further research is needed to evaluate the utility of LCT in clinical practice.