Mesenchymal stromal cells (MSCs) display robust reparative properties through their ability to limit apoptosis, enhance angiogenesis, and direct positive tissue remodeling. However, low in vivo survival of transplanted cells limits their overall effectiveness and significantly affects their clinical usage. Consequently, identifying strategies to improve cell survival in vivo are a priority. One explanation for their low survival is that MSCs are often transplanted into ischemic tissue, such as infarcted myocardium, where there is poor blood supply and low oxygen tension. Therefore, we examined how MSCs respond to a hypoxic, nutrient-poor stress environment to identify trophic factors that could be manipulated in advance of MSC transplantation. Combining microarray and proteomic screens we identified plasminogen activator inhibitor 1 (PAI-1) as one factor consistently upregulated in our in vitro ischemia-mimicking conditions. Subsequent genetic and chemical manipulation studies define PAI-1 as a negative regulator of MSC survival in vivo. Mechanistically, MSC-derived PAI-1 does not alter MSC survival through a plasmin-dependent mechanism but rather directly impacts on the adhesiveness of MSCs to their surrounding matrices. Thus we can conclude that post-transplantation, PAI-1 negatively impacts MSC survival by promoting anoikis via matrix detachment.