Abstract Ewing's sarcoma is the second most common bone cancer in adolescents and is characterized by the aberrant chimeric fusion gene EWS/FLI1. Wild-type EWS has been proposed to play a role in splicing and transcription, but how these functions affect early development is unknown. To elucidate the function of EWS in early development, we analyzed a mutant ewsa zebrafish line. We generated a maternal zygotic (MZ) ewsa/ewsa line because the ewsa/wt and ewsa/ewsa zebrafish appear to be normal and fertile. The adult MZ ewsa/ewsa mutants display defects in the craniofacial bones (dentary and basihyal). Compared to wt/wt, the MZ ewsa/ewsa mutants have a higher number of craniofacial prehypertrophic chondrocytes, and they fail to mature into hypertrophic chondrocytes possibly due to impaired intercalation at 4 days post-fertilization (dpf). The adult MZ ewsa/ewsa mutants also display curved spines due aberrant differentiation of the nucleus pulposus cells in the intervertebral discs (IVD). These altered differentiation is preceded by increased Collagen type II in the IVD, and Ctgf in the craniofacial chondrocytes in MZ ewsa/ewsa mutants compared to wt/wt at 36 hpf. We further discovered that both Ewsa and Ewsb interact with the master transcriptional factor for chondrogenesis, Sox9, in zebrafish. Consistently, qPCR analysis consistently demonstrated that Sox9 target genes are either upregulated (ctgfa, ctgfb, col2a1a, col2a1b) or downregulated (sox5, nog1, nog2, bmp4) in MZ ewsa/ewsa mutants compared to the wt/wt zebrafish embryos. This study is the first demonstration of Ewsa-dependent regulation of skeletogenesis through modulation of Sox9 target gene expression. Citation Format: Chris Merkes, Timothy K. Turkalo, Nicole Wilder, Hyewon Park, Mizuki Azuma. Ewing's sarcoma Ewsa protein regulates Sox9 during skeletogenesis in zebrafish. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 73. doi:10.1158/1538-7445.AM2014-73