Myelin pallor is frequently reported in HIV patients, and can occur in the CNS prior to other evidence of disease process. Our exploratory studies showed that oligodendrocytes (OLs) are direct targets of HIV-1 Tat (transactivator of transcription). Tat induces a dose-dependent increase of intracellular Ca2+ level ([Ca2+]i) in cultured murine OLs, which can be attenuated by ionotropic glutamate receptor (iGluR) antagonists MK801 and CNQX. The Tat-induced [Ca2+]i increase leads to increased death in immature (O4+, MBP-), but not mature (O4+, MBP+) OLs, over 96 h. In addition, Tat-induced [Ca2+]i increase also reduced myelin-like membrane production by mature OLs. Calcium/Calmodulin dependent kinase IIβ (CaMKIIβ) and glycogen synthase kinase 3β (GSK3β) have been known to regulate differentiation, myelination, and apoptosis in OLs. Since both CaMKIIβ and GSK3β are important downstream modulators of [Ca2+]i change, we hypothesized that the detrimental effects of Tat on immature/mature OL viability and function are mediated via CaMKIIβ and GSK3β activation. Our results showed that Tat activates both CaMKIIβ and GSK3β in immature OLs, but only activates CaMKIIβ in mature OLs. MK801 completely blocks Tat-induced CaMKIIβ and GSK3β activation in both immature and mature OLs, while CNQX blocks GSK3β activation, but has only a partial effect on CaMKIIβ activity. Blocking iGluRs or inhibiting GSK3β both rescue Tat-induced immature OL death, but only MK801 reverses the membrane injury in mature OLs. Together, these data strongly suggest that 1) activity of CaMKIIβ and GSK3β in OLs can be regulated by Tat-induced iGluRs activation and 2) OLs at different developmental stages show different responses to Tat, possibly due to activation of different signaling pathways.