PURPOSE: Vascularized composite allotransplantation (VCA) can include vascularized bone marrow (VBM) to potentially generate chimerism and subsequent tolerance in the recipient. While increasing ischemia time is known to be detrimental to free flaps and solid organ transplants, it is unknown whether increasing VCA ischemia time may diminish VBM’s capacity to achieve chimerism and tolerance. This study determines whether increasing cold ischemia time results in decreased peripheral chimerism levels, increasing loss of the donor bone marrow compartment, and increased skin paddle rejection in a murine VCA model. METHODS: Twenty-three adult Lewis rats received osteomyocutaneous hind limb allografts from 3 groups of Brown-Norway rats. The groups underwent 0, 6, and 12 hours of cold ischemia at 4°C (n=8, 6, and 9 respectively). Immunosuppressive conditioning consisted of anti-lymphocyte serum 1cc 3 days pre-operatively and on post-op day (POD) 1, tacrolimus 2mg/kg/day for PODs 1–7, and then concluded with rapamycin 3mg/kg/day for PODs 8–28. Peripheral chimerism levels were drawn after POD 30. Skin paddle was observed for 30 days after immunosuppression cessation for rejection or tolerance. Allograft was collected at POD 60 for flow cytometry and histologic analysis. RESULTS: Peripheral chimerism levels in both myeloid and lymphoid lines were decreased in the 12 & 6 hours groups versus 0 hours (p<0.01). At POD 60, the amount of donor cells in the allograft bone marrow was significantly decreased at 12 & 6 hours versus the 0 hours group (p<0.05). At 12 hours, hematoxylin & eosin histology demonstrated significantly increased reactive lymphocytes, increased stroma, significantly decreased hematopoetic cells, and significant myelofibrosis. At 6 hours, moderate stromal increases, moderately decreased hematopoetic cells, and moderate myelofibrosis was observed. Trichrome staining revealed viable bone at all time points though with there were increasing amounts of collagen deposition with increasing ischemia and osteoslerosis was noted at 12 hours. Microvasculature was preserved at all time points. By POD 60, 0% of skin paddles survived in the 12 hours group, 17% at 6 hours, and 73% at 0 hours. CONCLUSION: With increasing ischemia time prior to VCA re-vascularization, there is decreased peripheral chimerism levels, increased loss of donor hematopoetic cells in the donor bone marrow, and increased skin paddle rejection. These findings indicate that ischemia time may significantly limit the capacity for VBM to facilitate chimerism and subsequent VCA tolerance and thus strategies using VBM to induce tolerance may need to consider the additional obstacle of time. Prolonged ischemia time may be a contributing factor to the absence of stable chimerism and tolerance in human VBM-bearing VCA recipients.