Ischemic preconditioning (IPC) is a potent and effective means of protecting cells against ischemic injury. The protection has been demonstrated to involve release of paracrine factors that promote cell survival and angiogenesis, factors important for successful tissue engineering. The aim of the present study was to determine whether IPC of a vascular bed in vivo is an effective strategy to prepare it for tissue engineering with implanted cells. To test this hypothesis, an in vivo vascularized tissue engineering approach was employed, whereby polyacrylic chambers were placed around the femoral vessels of adult Sprague-Dawley rats. IPC was induced by 3 cycles of 5 min femoral artery occlusion interspersed with 5-min periods of reperfusion. Rats subjected to IPC generated bigger tissue constructs at 7 and 28 days postimplantation of empty chambers (∼50% increase in weight and volume, p<0.05). Morphometric counting of Masson trichrome stained tissue sections revealed significantly greater tissue construct volumes in ischemic preconditioned vascular beds at 7 and 28 days, increasing both fibrin matrix and vascularized tissue. Furthermore, morphometry of lectin-labeled blood vessels indicated an increase in vascular volume in IPC tissue constructs (∼100% increase vs. control, p<0.05). To investigate the cytoprotective effect of IPC, we implanted DiI-labeled neonatal rat cardiomyocytes in the chambers for 3 days, and IPC significantly reduced apoptosis of implanted cells as determined by the TUNEL assay and cleaved caspase-3 immunostaining. Furthermore, IPC significantly increased the cardiac muscle volume and vascular volume at 28 days after implantation of cardiomyocytes. In conclusion, in vivo IPC promotes survival of implanted cardiomyocytes and is associated with enhanced angiogenesis. IPC may represent a new approach to optimize tissue engineering with implanted cells.