We have developed a potent, histone deacetylase 8 (HDAC8)-specific inhibitor PCI-34051 with >200-fold selectivity over the other HDAC isoforms. PCI-34051 induces caspase-dependent apoptosis in cell lines derived from T-cell lymphomas or leukemias, but not in other hematopoietic or solid tumor lines. Unlike broad-spectrum HDAC inhibitors, PCI-34051 does not cause detectable histone or tubulin acetylation. Cells defective in T-cell receptor signaling were still sensitive to PCI-34051-induced apoptosis, whereas a phospholipase C-gamma1 (PLCgamma1)-defective line was resistant. Jurkat cells showed a dose-dependent decrease in PCI-34051-induced apoptosis upon treatment with a PLC inhibitor U73122, but not with an inactive analog. We found that rapid intracellular calcium mobilization from endoplasmic reticulum (ER) and later cytochrome c release from mitochondria are essential for the apoptotic mechanism. The rapid Ca(2+) flux was dependent on PCI-34051 concentration, and was blocked by the PLC inhibitor U73122. Further, apoptosis was blocked by Ca(2+) chelators (BAPTA) and enhanced by Ca(2+) effectors (thapsigargin), supporting this model. These studies show that HDAC8-selective inhibitors have a unique mechanism of action involving PLCgamma1 activation and calcium-induced apoptosis, and could offer benefits including a greater therapeutic index for treating T-cell malignancies.