The ability to discriminate cell adhesion molecule expression between healthy and inflamed endothelium is critical for therapeutic intervention in many diseases. This study explores the effect of laminar flow on TNF-induced E-selectin surface expression levels in human umbilical vein endothelial cells (HUVECs) relative to IL-1-induced expression via flow chamber assays. HUVECs grown in static culture were either directly (naive) activated with cytokine in the presence of laminar shear or pre-exposed to 12h of laminar shear (shear-conditioned) prior to simultaneous shear and cytokine activation. Naive cells activated with cytokine in static served as control. Depending on the cell shear history, fluid shear is found to differently affect TNF-induced relative to IL-1-induced HUVEC expression of E-selectin. Specifically, E-selectin surface expression by naive HUVECs is enhanced in the 812h activation time range with simultaneous exposure to shear and TNF (shear-TNF) relative to TNF static control whereas enhanced E-selectin expression is observed in the 424h range for shear-IL-1 treatment relative to IL-1 static control. While exposure of HUVECs to shear preconditioning mutes shear-TNF-induced E-selectin expression, it enhances or down-regulates shear-IL-1-induced expression dependent on the activation period. Under dual-cytokine-shear conditions, IL-1 signaling dominates. Overall, a better understanding of E-selectin expression pattern by human ECs relative to the combined interaction of cytokines, shear profile and history can help elucidate many disease pathologies. Biotechnol. Bioeng. 2013; 110: 9991003. (c) 2012 Wiley Periodicals, Inc.