Histone deacetylase inhibitors (HDACi) have been reported to increase tumor antigen expression, and have been successfully tested as adjuvants for melanoma immunotherapy in mouse models. In this work, we tested the effects of a pan-HDACi on human lymphocytes and melanoma cell lines. Effects of the pan-HDACi panobinostat (LBH589) on cell viability, cell cycle, apoptosis, and DNA damage were determined in peripheral blood mononuclear cells (PBMC) from 2 healthy donors, 13 patients with metastatic melanoma, 2 bone marrow samples from patients with different malignances, and 12 human melanoma cell lines. Intracellular signaling in lymphocytes, with or without cytokine stimulation, was analyzed by phospho-flow cytometry in one of each type. The IC50 in PBMCs was <20 nmol/L compared with >600 nmol/L in melanoma cell lines; >40% apoptotic cell death in PBMCs versus <10% in melanoma cell lines was seen at the same concentration. Phospho-histone variant H2A.X (pH2A.X) increased 2-fold in healthy donor PBMCs at 1 nmol/L, whereas the same effect in the melanoma cell line M229 required 10 nmol/L. pH2A.X was inhibited slightly in the PBMCs of 3 patients with metastatic melanoma at 1 nmol/L and in the melanoma cell line M370 at 10 nmol/L. Panobinostat inhibited phospho-STAT1/3/5/6, -p38, -ERK, -p53, -cyclin D3, and -histone H3 in flow cytometry-gated healthy donor B and T cells, whereas it induced up to 6-fold activation in patients with metastatic melanoma and bone marrow samples. In human lymphocytes, panobinostat alters key lymphocyte activation signaling pathways and is cytotoxic at concentrations much lower than those required for melanoma antitumor activity, resulting in an adverse therapeutic window.