It has been demonstrated that restoration of function to compromised tissue can be accomplished by transplantation of bone marrow stem cells and/or embryonic stem cells (ESCs). One limitation to this approach has been the lack of noninvasive techniques to longitudinally monitor stem cell attachment and proliferation. Recently, murine ESC lines that express green fluorescent protein (GFP), luciferase (LV), and herpes simplex thymidine kinase (HVTK) were developed for detection of actively growing cells in vivo by imaging. In this study, the authors investigated the use of these ESC lines in a burned mouse model using Integra® as a delivery scaffolding/matrix. Two different cell lines were used: one expressing GFP and LV and the other expressing GFP, LV, and HVTK. Burn wounds were produced by application of a brass block (2 × 2 cm kept in boiling water before application) to the dorsal surface of SV129 mice for 10 seconds. Twenty-four hours after injury, Integra® with adherent stem cells was engrafted onto a burn wound immediately after excision of eschar. The stem cells were monitored in vivo by measuring bioluminescence with a charge-coupled device camera and immunocytochemistry of excised tissue. Bioluminescence progressively increased in intensity over the time course of the study, and GFP-positive cells growing into the Integra® were detected. These studies demonstrate the feasibility of using Integra® as a scaffolding, or matrix, for the delivery of stem cells to burn wounds as well as the utility of bioluminescence for monitoring in vivo cellular tracking of stably transfected ESC cells.