Cell therapy with mesenchymal stromal cells (MSCs) was found to protect neurons from damage after experimental stroke and is currently under investigation in clinical stroke trials. In order to elucidate the mechanisms of MSC-induced neuroprotection, we used the in vitro oxygen–glucose deprivation (OGD) model of cerebral ischemia. Co-culture of primary cortical neurons with MSCs in a transwell co-culture system for 48 h prior to OGD-reduced neuronal cell death by 30-35%. Similar protection from apoptosis was observed with MSC-conditioned media when added 48 h or 30 min prior to OGD, or even after OGD. Western blot analysis revealed increased phosphorylation of STAT3 and Akt in neuronal cultures after treatment with MSC-conditioned media. Inhibition of the PI3K/Akt pathway completely abolished the neuroprotective potential of MSC-conditioned media, suggesting that MSCs can improve neuronal survival by an Akt-dependent anti-apoptotic signaling cascade. Using mass spectrometry, we identified plasminogen activator inhibitor-1 as an active compound in MSC-conditioned media. Thus, paracrine factors secreted by MSCs protect neurons from apoptotic cell death in the OGD model of cerebral ischemia.