Human embryonic stem cell-derived endothelial cells (hESC-EC) are being investigated as research tools and cell therapy for vascular inflammation. We have shown that hESC-EC do not respond to certain bacteria pathogen associated molecular patterns (PAMP). Endothelial cells also respond to viral PAMPs via toll-like receptor-3 (TLR3) which couples with IRF transcription factors. IRF can also be activated by interferons (IFN) which are also released during immune reactions. We have compared PolyIC (10 μg/ml; TLR3 agonist), and IFN (30 ng/ml) responses in hESC-EC and human umbilical vein endothelial cells (HUVEC). CXCL8 and IP10 were measured by ELISA as biomarkers for NF-κB and IRF pathways respectively. All data are % control (100) ± SEM n=5–6. Both hESC-EC (IFNα; 289.8±71.6, IFNβ; 656.8±182.3, IFNγ; 878.6±275.9, IFNλ 55.1±18.7) and HUVEC (IFNα; 1419.3±445.8, IFNβ; 3144.8±946.6, IFNγ; 2982.7±583.9, IFNλ; 132.8±30.1) released IP10 in response to each IFN (p<0.05; one-sample t test) except IFNλ (p>0.05; one-sample t test). HUVEC (3593.4±1269.4) but not hESC-EC (73.3±12.0) also responded to PolyIC with release of IP10. CXCL8 was not elevated by any treatment in either cell type. hESC-EC and HUVEC have a functional IRF transcription factor pathway that is activated in response IFNα, β and γ but not IFNλ. hESC-EC do not have a detectable TLR3 function. These findings may have implications for the use of hESC-EC in research and therapy and a more thorough investigation of these pathways is needed.