Intestinal ischemia after trauma/hemorrhagic shock (T/HS) results in gut barrier dysfunction and the production/release of biologically active and tissue injurious factors in the mesenteric lymph which in turn causes acute lung injury and a systemic inflammatory state. Since T/HS-induced lung injury is associated with pulmonary endothelial and epithelial programmed cell death (PCD) and was abrogated by mesenteric lymph duct ligation, we sought to investigate the cellular pathways involved. Compared with trauma sham shocked (T/SS) rats, a significant increase in caspase-3 and M30 expression was detected in the pulmonary epithelial cells undergoing PCD, whereas apoptosis inducing factor (AIF), but not caspase-3, was detected in endothelial cells undergoing PCD. This AIF-mediated pulmonary endothelial PCD response was validated in an in situ femoral vein assay where endothelial cells were found to express AIF but not caspase-3. To complement these studies, human endothelial cells (HUVEC, HLMEC), and the alveolar epithelial cells (A549) were used as in vitro models. T/HS lymph induced the nuclear translocation of AIF in HUVEC and HLMEC cells and caspase inhibition in these cells did not afford any cytoprotection. For proof of principle, AIF silencing in HUVEC cells reversed the cytotoxic effects of T/HS on cell viability and DNA fragmentation. In A549 cells, T/HS lymph activated caspase-3 mediated apoptosis which was partially abrogated by zVAD. Additionally, T/HS lymph did not cause the nuclear translocation of AIF in A549 cells. Collectively, T/HS induced pulmonary endothelial PCD occurs via an AIF-dependent caspase independent pathway while epithelial cells undergo apoptosis by a caspase-dependent pathway.