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Ewing sarcoma (EwS) is driven by the EWS-FLI1 (EF) oncoprotein and is assumed to originate from mesenchymal progenitors during bone development. Attempts to generate a EwS genetic mouse model by targeting EF expression to the mesenchymal lineage so far failed to result in tumorigenesis. Here, we investigated the influence of bone niche-derived factors on EF dependent transformation. We focused on IGF1 which is highly enriched in the bone during puberty when EwS incidence peaks, and which was previously proposed to play an essential role in EwS pathogenesis. We restricted EF expression to mesenchymal stem cells during endochondral bone formation by crossing an EF knock-in mouse with a mouse expressing Prx1-driven Cre recombinase. The resulting EFprx1 progeny died after birth as consequence of severe skeletal malformations resulting from differentiation arrest of early chondrocytes. In vitro, these embryonic EF transgenic chondrocyte-like mesenchymal stem cells (EFprx1 MSCLC) are immortal, but largely failed to form colonies in soft-agar as functional surrogate of full malignant transformation. In contrast, when treated with IGF1 for 4 weeks, a significant increase in soft-agar colony formation was observed, which was dependent on sustained EF expression. Strikingly, cells from these colonies maintained their transformed phenotype in absence of further IGF1 treatment and gave rise to aggressively growing tumors upon xenotransplantation in mice. Analysis of differentially expressed genes from these stably IGF1-reprogrammed, fully transformed EFprx1 MSCLC showed a significant enrichment for the EwS specific EF transcriptional signature with upregulation of Foxm1 and cell cycle-related pathways, and down-regulation of apoptotic gene sets. Cluster analysis of open chromatin regions in EF immortalized versus fully transformed and wildtype cells revealed five distinct chromatin modules. Of these, module 2 comprises genomic regions that open up exclusively in IGF1 reprogrammed EF expressing cells. Among associated genes, Yap1 was most highly induced in IGF1 reprogrammed EFprx1 MSCLC. Consistent with this finding, RNA-seq revealed enrichment of a Yap1 transcriptional signature in IGF1 reprogrammed versus parental EF expressing cells. In addition, chromatin module 4, which comprises regions open in parental and IGF1 reprogrammed EFprx1 MSCLC but not in wildtype MSC, is highly enriched in binding motifs for TEAD transcription factors, the functional effectors of YAP1 signaling. We therefore hypothesize that YAP1 induction by IGF1 reprogramming synergizes with EF in the activation of module 4 genes. Strikingly, modules 2 and 4 comprise hallmark genes of stemness and self-renewal which also characterize human EwS, including Bcl11b, Prkcb, Sox2, Ccnd1, and Klf5, as well as the top EwS dependency gene Igf2bp1. Our data suggest that transient IGF1 exposure in the bone microenvironment during puberty may be required for EF driven EwS tumor initiation, and we identify a central role of YAP1 in the early phases of EwS pathogenesis. Citation Format: Heinrich Kovar, Rahil Noorizadeh, Florian Halbritter, Utkarsh Kapoor, Wolfgang Mikulits, Richard Moriggl, Barbara Sax. YAP1 is a key regulator of EWS-FLI1-dependent malignant transformation upon IGF1 mediated reprogramming of bone mesenchymal stem cell-like cells [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr PR003.