Purpose: To investigate the outcome of a new technique of ex vivo expanded stem cell allograft for limbal stem cell deficiency (LSCD), and to characterize the ocular surface genotype after surgery. Design: Retrospective noncomparative case series. Participants: Ten eyes of 10 patients with profound LSCD arising from ectodermal dysplasia (3 eyes), Stevens–Johnson syndrome (3 eyes), chemical injury (2 eyes), thermal injury (1 eye), and rosacea blepharoconjunctivitis (1 eye). Intervention: Allogeneic corneal limbal stem cells were cultured on plastic and transplanted to the recipient eye after removal of conjunctival pannus. Amniotic membrane was applied in a bandage capacity. The procedure was combined with other reconstructive surgery in 2 cases. Nine patients received systemic cyclosporin A immunosuppression, and the DNA genotype was investigated with surface impression cytology. Main outcome measures: Parameters of LSCD, including vascularization, conjunctivalization, inflammation, epithelial defect, photophobia, and pain. Results: The mean follow-up period was 28 months (range, 12–50). Seven of 10 eyes (70%) had improved parameters of LSCD at final follow-up and were considered successes. Four (40%) had improved visual acuity, including 3 having had further procedures for visual rehabilitation. Three patients failed to improve—1 with a thermal burn and lid deformity, 1 with Stevens–Johnson syndrome and severe dry eye, and 1 with ectodermal dysplasia who developed an epithelial defect at 26 months. DNA analysis of the first 7 cases showed no ex vivo donor stem cell DNA present beyond 9 months. Conclusions: Ex vivo expanded stem cell allograft is a useful technique for restoring the ocular surface in profound LSCD. The absence of donor DNA beyond 9 months suggests that ongoing immunosuppression may be unnecessary and raises questions regarding the origin of the host corneal epithelium.