Despite combined antiretroviral (ARV) therapy (cART), human immunodeficiency virus (HIV)-associated neurocognitive disorder (HAND) occurs in 30-50% of HIV+ patients and include white matter (WM) and myelin pathology. These pathologies persist in HAND patients regardless of effective viral control through cART. cART-treated HAND patients exhibit thinning of the corpus callosum and disruption of WM microstructures. Despite many studies recognizing the persistence of WM loss, mechanisms regulating these abnormalities and their consequences have not been studied. Taken together, we hypothesized that HIV infection and/or cART could alter oligodendrocyte (OL) maturation, function and/or survival, influencing the persistence of WM pathologies in HAND patients in the post-cART era. We utilized a well-established primary rat OL precursor cell (OPC) culture model to examine the effect of HIV infection or select ARV compounds on OL maturation and survival. To model the effect of HIV on OL maturation, we generated HIV-infected monocyte derived macrophage supernatants (HIV/MDMs) to mimic the neuroinflammatory HIV-infected CNS environment. Separately, we investigated the effect of select ARV drugs: elvitegravir (EVG), raltegravir (RAL), darunavir (DRV) and saquinavir (SQV) on OL maturation. At the time of differentiation, OPCs were treated with either HIV/MDMs or select ARV agents and allowed to mature for 72 hours. Immunocytochemistry was used to examine specific OL lineage proteins and cell death. Immunoblotting was used to assess expression of myelin proteins, proteolipid protein (PLP) and myelin basic protein (MBP). Based on previous evidence from our group and others, we used specific pharmacological inhibitors to determine whether glutamate signaling, the integrated stress response (ISR) and/or lysosome dysfunction mediated the inhibitory effect of HIV/MDMs or ARV compounds on OL maturation. Treatment of OPCs with HIV/MDMs, EVG, DRV, or SQV all separately inhibited OL maturation, in vitro while treatment with RAL had no effect. HIV/MDMs, EVG, DRV or SQV all inhibited expression of PLP and MBP while RAL had no effect. We observed that AMPA/Kainate receptors and the ISR mediated the effect of HIV/MDMs on OL maturation. The ISR also mediated EVG-induced inhibition of OL maturation. Finally, we demonstrated that DRV and SQV decrease the number of acidic lysosomes in OLs and OL maturation was rescued following reacidification of lysosomes. These studies demonstrate that glutamate signaling, the ISR and lysosome dysfunction can mediate HIV/MDMs- or ARV drug-driven inhibition of OL maturation. These pathways might be potential therapeutic mechanisms used adjunctively with cART to ameliorate WM pathology in HAND patients with further need to investigate their contribution to cognitive impairment.