Emerging studies indicate that split thickness skin grafting is an effective treatment against sulfur mustard injury. We are developing bioengineered skin from mouse embryonic stem cells for the treatment of cutaneous vesicant injuries. Mouse embryonic stem cells were differentiated into keratinocytes in a membrane filter using stem cell culture medium containing ascorbic acid. The differentiated keratinocytes were subjected to air/liquid culture to develop the bioengineered skin. The formation of skin samples were assessed by immunofluorescence microscopy using layer specific antibodies including basal layer skin markers cytokeratin 14, fibronectin, and collagen type IV. The bioengineered skin was frozen in 10% DMSO at -80oC over night and then transferred to liquid nitrogen storage. Therapeutic efficacy of the bioengineered skin was evaluated in a C57BL/6 black mouse back model of vesicant injury using 3 ml of CEES and an exposure of 10 min. Subsequent to CEES exposure at 96 h, the injury site was cleaned with debridase, but the treatment did not remove the injured skin layer. Early application of debridase produced more severe injury, most likely due to the additional proteolytic activity. Therapy with bioengineered skin after excising the injured skin and debridase treatment showed skin growth and healing in 1 to 3 weeks. The CEES control animals showed no skin growth but self healing of the injured area by contraction of the skin. Cryopreservation methods for bioengineered skin are currently being established for field applications, and optimal conditions are being assessed by Trypan blue viability and MTT cytotoxicity assays of the frozen and then thawed skin cells.