Pluripotent stem cells (PSCs) have revolutionized in vitro approaches for disease modeling and regenerative medicine. Nowadays, avian stem cells represent also a tremendous alternative to primary cells or eggs for the study of viruses and for the production of vaccines. They offer the possibility of developing stable production methods while preventing safety risks and egg shortages due to the increasing number of epizootics in the poultry farms. However, so far, only chicken embryonic stem cells (ESC) lines and one proprietary duck ESC line have been established and all of them were derived in the presence of serum. Therefore, we sought to establish novel duck ESCs in chemically defined media and analyzed their permissiveness to influenza viruses to provide a novel substrate for viral replications. Methods: Derivation and characterization of duck ESCs: Pekin duck blastodermal cells from freshly laid eggs were cultured in chemically defined media and different independent isolates were established for long term culture. They were characterized by biochemical assays (Telomerase and alkaline phosphatase activities). Their transcriptomes were analyzed by RT-qCR and RNAseq, and their plasticity was evaluated by in vitro differentiation and by chimaera formation. Influenza infections: A/Lyon/969/2009 H1N1 (H1N1pdm09), A/Puerto Rico/8/1934 (H1N1), A/Switzerland/ 9715293/2013 (H3N2), B/Phuket/3073/2013 and A/Finch/England/2051/1991 (H5N2) (low pathogenic avian influenza virus) were propagated in the MDCK cells at 37°C in Eagle’s minimum essential medium (EMEM Lonza) supplemented with 1 µg/mL TPCK-treated trypsin (Sigma-Aldrich, St. Louis, MO, USA). The first HPAIV (High Pathogenicity Avian Influenza Virus) H5N8 was generated by reverse genetic using the eight segments from the A/mallard duck/France/171201g/2017 (H5N8) as described by Bessiere et al, 2022. H5N1/2021 and H5N8/2020 were isolated from field samples (feathers). All those three HPAIV were amplified on 9 to 11 days embryonated eggs. The allantoic liquid was harvested after 48 hours of infection, aliquoted and frozen at -70°C. The infectious viral titers were determined either by 50% tissue culture infectious dose (TCID50) in the MDCK cells from 4 replicates using the Reed and Muench method. Results : Independent isolates were established from duck embryos in chemical defined media without serum. We demonstrated that these cells exhibit a typical transcriptional signature of ESCs with high OCT4 and NANOG gene expressions but also express high level of germinal markers such as DDX4 and DAZL genes. These cells display some physiological markers of ESC, especially cellular plasticity demonstrated by in vitro differentiation and by their ability to colonize developing embryo and form chimaeras. These populations then share most of the characteristics expected of pluripotent stem cells and can provide a working basis to analyze the mechanisms of maintenance of pluripotency in avian species by comparing them with already existing chicken cells. Once established we first checked the expression level of endogenous retroviral elements (ERV), which are usually silent in most cell types, but which can be transcriptionally reactivated in pluripotent cells. Therefore, we analyzed the activity of reverse transcriptase activity in the supernatant of those new duck ESCs by qPERT assay. We demonstrated that those cells exhibit a much lower RT activity than their chicken ESCs homologs and could therefore be viewed as novel valuable substrates for viral studies. We then infected both chicken and duck ESCs with 6 different influenza viruses (H1N1/pr8, H1N1/pdm2009, H5N2, H5N1/2021, H5N8/2017 and H5N8/2020) and evaluated the cells permissiveness either by measuring the Influenza infectious particles in the supernatant by TCID50 on MDCK cells or by measuring the Influenza viral RNA load in supernatant by RT-qPCR using primers targeting the HA gene. We demonstrated that these new duck ESCs but not duck embryonic fibroblasts, can replicate very efficiently avian influenza viruses when compared to chicken ESCs and other reference cell lines. Conclusions and Perspectives: Therefore, all these properties encompassed in these new duck stem cell lines (long-term cultivation, permissiveness towards avian influenza viruses and low retroviral activity) make these cells a perfect substrate for evaluating new influenza isolates from the field as well as for producing vaccines under greater safety conditions when compared to chicken embryos.