Chronic exposure to cigarette smoke can lead to endothelial dysfunction and potentially endothelial cell death. Here, we exposed Human Aortic Endothelial Cells (HAECs) to whole smoke conditioned media (WSCM) over a range of nicotine equivalence (n.e.) concentrations (0-8000 ng/mL n.e.). After 24 h, Neutral Red Uptake (NRU) and reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to formazan was determined for each exposure concentration and compared to control. IC50 values in the NRU assay were: 4582 ng/mL n.e. ± 1074, 4587 ng/mL n.e. ± 951, 4993 ng/mL n.e. ± 1239 and 4691 ng/mL n.e. ± 402 for four HAEC donors. IC50 values in the MTT assay were: 4885 ng/mL n.e. ± 1341, 4584 ng/mL n.e. ± 806, 5749 ng/mL n.e. ± 783 and 5228 ng/mL n.e. ± 593 for the four donors. To examine the mechanism responsible for WSCM-induced cytotoxicity in HAECs, flow cytometry using necrosis (Propidium Iodide) and apoptosis (Annexin V) markers were used. Annexin V-positive cell populations increased in a dose dependent manner while increases in PI-positive cell populations occurred at the highest doses of WSCM (5000-8000 ng/mL n.e.). Western blotting for cleaved caspase-3 confirmed that apoptosis occurs at >5000 ng/mL n.e. WSCM, coinciding with reduced HAEC survival.