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The mechanisms of infection and dissemination remain poorly understood. Thus, elucidating how USUV spreads in a susceptible host is crucial for identifying therapeutic targets. To investigate host defenses against USUV, we generated an infectious clone of the TC508 isolate. After characterizing its replication dynamics in cultured cells from multiple species, we investigated its pathogenesis in an array of mice with genetic perturbations. Previous studies demonstrated that whole-body deletion of type I interferon (IFN) signaling led to widespread USUV infection and fatality in mice. Here, we observed the same lethal phenotype in STAT1-deficient mice and identified hematopoietic cells specifically as central to USUV pathogenesis in a mammalian host. Deletion of STAT1 in all hematopoietic subsets, but not hepatocytes, neurons, macrophages or conventional dendritic cells, was sufficient for systemic viral dissemination and ultimate fatality. Conversely, mice lacking functional B, T, and natural killer (NK) cells but with intact myeloid cells were resistant to USUV. Our findings provide new insights into the tissue-specific barriers that regulate USUV infection and underscore the importance of innate immunity in host defense for this important emerging flavivirus.<\/jats:p>","DOI":"10.1371\/journal.pntd.0013317","type":"journal-article","created":{"date-parts":[[2025,7,22]],"date-time":"2025-07-22T17:32:01Z","timestamp":1753205521000},"page":"e0013317","update-policy":"https:\/\/doi.org\/10.1371\/journal.pntd.corrections_policy","source":"Crossref","is-referenced-by-count":1,"title":["STAT1-mediated interferon signaling in the hematopoietic system is essential for restricting Usutu virus infection in vivo"],"prefix":"10.1371","volume":"19","author":[{"given":"Amy N.","family":"Nelson","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Saloni","family":"Sinha","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sydney 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