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Kaposi's Sarcoma herpesvirus (KSHV) is the etiologic agent of Kaposi's Sarcoma (KS), a highly vascularized tumor common in AIDS patients and many countries in Africa. KSHV is predominantly in the latent state in the main KS tumor cell, the spindle cell, a cell expressing endothelial cell markers. To identify host genes important for KSHV latent infection of endothelial cells we previously used a global CRISPR/Cas9 screen to identify genes necessary for the survival or proliferation of latently infected cells. In this study we rescreened top hits and found that the highest scoring gene necessary for infected cell survival is the anti-apoptotic Bcl-2 family member Bcl-xL. Knockout of Bcl-xL or treatment with a Bcl-xL inhibitor leads to high levels of cell death in latently infected endothelial cells but not their mock counterparts. Cell death occurs through apoptosis as shown by increased PARP cleavage and activation of caspase-3/7. Knockout of the pro-apoptotic protein, Bax, eliminates the requirement for Bcl-xL. Interestingly, neither Bcl-2 nor Mcl-1, related and often redundant anti-apoptotic proteins of the Bcl-2 protein family, are necessary for the survival of latently infected endothelial cells, likely due to their lack of expression in all the endothelial cell types we have examined. Bcl-xL is not required for the survival of latently infected primary effusion lymphoma (PEL) cells or other cell types tested. Expression of the KSHV major latent locus alone in the absence of KSHV infection led to sensitivity to the absence of Bcl-xL, indicating that viral gene expression from the latent locus induces intrinsic apoptosis leading to the requirement for Bcl-xL in endothelial cells. The critical requirement of Bcl-xL during KSHV latency makes it an intriguing therapeutic target for KS tumors. Author summary: KSHV is the cause of Kaposi's Sarcoma, an endothelial cell-based malignancy that largely affects individuals with HIV-AIDS and individuals in many countries in sub-Saharan Africa. There are currently no direct treatments for KSHV and therefore, novel therapeutic targets are needed. To address this, we utilized a screen to identify cellular genes required for the survival of infected endothelial cells. We found that Bcl-xL, but not other anti-apoptotic proteins in the same family, is necessary for the survival of KSHV latently infected endothelial cells, but not uninfected cells and that KSHV latent gene expression induces intrinsic apoptosis in this cell type. Further, no other cell types we tested required Bcl-xL for survival. Thus, endothelial cells uniquely require Bcl-xL for survival during KSHV latent infection. With several Bcl-xL inhibitors currently undergoing clinical trials, these findings raise the potential to target KS tumors through inhibition of Bcl-xL. [ABSTRACT FROM AUTHOR]