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Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma and can be divided into subtypes based on the primary genetic driver. One of the most lethal subtypes is fusion-positive RMS, which is most commonly driven by chimeric oncogene PAX3::FOXO1. While PAX3::FOXO1’s full function is still unknown, this fusion is known to act as a novel transcription factor and epigenetic modifier, dysregulating expression of a multitude of downstream targets. Notably, one of its direct targets is fibroblast growth factor receptor 4 (FGFR4). FGFR4 plays a significant role in myogenesis, and other groups have shown it is activated or overexpressed in RMS and multiple other cancer types. Specifically, in rhabdomyosarcoma, FGFR4 is overexpressed in the fusion-positive subtype in comparison to the fusion-negative subtype, and its overexpression is correlated with reduced overall survival. As such, FGFR4 is being pursued as a potential therapeutic target. Here, we aim to establish a stable fgfr4 knockout zebrafish line to investigate the in vivo genetic cooperation of PAX3::FOXO1 and fgfr4. We hypothesize that the loss of fgfr4 will reduce PAX3::FOXO1-mediated tumorigenesis. To generate fgfr4 knockout mutants, we injected single-cell zebrafish embryos with Cas9 protein and fgfr4 guide RNA, identified adult founders, and outcrossed to wildtype zebrafish to create an F1 generation. These F1 fish were analyzed using high resolution melt analysis, and fish with putative mutations were sequenced to determine the exact mutation. We have validated that the maternal zygotic knockout fish are indeed null for Fgfr4 mRNA using qRT-PCR. Currently, will are incorporating the knockout line with our transgenic human PAX3::FOXO1 zebrafish tumor models to study fgfr4’s direct impact on PAX3::FOXO1- mediated mechanisms of tumorigenesis. These studies, along with the use of other zebrafish and cell culture models developed by our lab, will generate new data on PAX3::FOXO1’s genetic dependency on FGFR4 function for in vivo activity and tumorigenesis.