Introduction: The use of hematopoietic cell transplantation (HCT) has previously been shown to ameliorate cutaneous blistering in pediatric patients with recessive dystrophic epidermolysis bullosa (RDEB), an inherited skin disorder that results from loss-of-function mutations in COL7A1 and manifests as deficient or absent type VII collagen protein (C7) within the epidermal basement membrane. Mesenchymal stem cells (MSCs) found within the HCT graft are believed to be partially responsible for this amelioration, in part due to their intrinsic immunomodulatory and trophic properties and also because they have been shown to restore C7 protein following intradermal injections in models of RDEB. However, MSCs have not yet been demonstrated to improve disease severity as a stand-alone systemic infusion therapy. Improving the efficacy and functional utility of MSCs via a pre-transplant conditioning regimen may bring systemic MSC infusions closer to clinical practice. Methods: MSCs were isolated from 2- to 4-week-old mice and treated with varying concentrations of transforming growth factor-β (TGFβ; 5-20 ng/mL), tumor necrosis factor- α (TNFα; 10-40 ng/mL), and stromal cell-derived factor 1-α(SDF-1α; 30 ng/mL) for 24-72 hours. Results: We demonstrate that treating murine MSCs with exogenous TGFβ (15 ng/mL) and TNFα (30 ng/mL) for 48 hours induces an 8-fold increase in Col7a1 expression and a significant increase in secretion of C7 protein, and that the effects of these cytokines are both time and concentration dependent. This cytokine treatment also promotes a 4-fold increase in Tsg-6 expression, a gene whose product is associated with improved wound-healing and immunosuppressive features. Finally, the addition of exogenous SDF-1α to this regimen induces a simultaneous upregulation of Col7a1, Tsg-6, and Cxcr4 expression. Conclusions: These data suggest that preconditioning represents a feasible method for improving the functional utility of MSCs in the context of RDEB stem cell transplantation, and also highlight the applicability of preconditioning principles toward other cell-based therapies aimed at treating RDEB patients.