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In multiple myeloma (MM), Notch signaling, a pathway mediating cell-to-cell communication between cells in the tumor niche, promotes MM proliferation and bone destruction. We previously reported that osteocytes (Ots), the most abundant bone cells, provide a microenvironment conducive for MM progression and bone destruction. Ots activate Notch signaling, increase Notch3 expression, and stimulate proliferation in MM cells. We detected Notch3 in CD138+ cells from MM patients and human and murine MM cell lines. Yet, the role of Notch3 in MM is unknown. Here, we used a shRNA approach to knockdown Notch3 to study its role in MM cells and their communication with Ots. The protein levels of active Notch3 (NICD3) were decreased by 80% in 5TGM1 MM cells transduced with shRNA against Notch3 compared to control shRNA-control cells. In contrast, Notch1, 2, and 4 NICD protein levels remained unchanged compared to controls. Notch3 knockdown decreased Notch target gene and cyclinD1 expression, reduced proliferation by 35%, and modestly increased apoptosis in MM cells. Additionally, Notch3 knockdown decreased Rankl expression and the ability of MM cells to promote osteoclastogenesis in vitro. Consistent with these observations, bioinformatic analysis of the transcriptome of CD138+ cells from newly diagnosed patients revealed upregulated processes related to positive regulation of cell proliferation and osteoclastogenesis in MM patients with high vs. low Notch3 expression. Next, we examined the role of Notch3 in MM-Ots communication. Notch3 knockdown in MM cells partially prevented the upregulation of Notch target genes and cyclinD1 expression and proliferation induced by direct contact with Ots. Inhibition of all Notch receptors with GSI fully prevented osteocyte-induced proliferation and Notch activation, suggesting that in addition to Notch3, other Notch receptors mediate MM-osteocyte communication. Remarkably, shRNA-mediated Notch2 inhibition did not alter MM cell proliferation or communication with osteocytes. Lastly, we analyzed the effects of Notch3 knockdown in MM cells in ex vivo and in vivo models. Using ex vivo bone organ cultures, we found less MM proliferation and lower levels of the resorption marker CTX in conditioned media from bones cultured with shRNA-Notch3 MM cells compared to control bones cultured alone. We injected mice intratibially with shRNA-Notch3 or shRNA-control 5TGM1 MM cells. After 5 weeks, mice bearing shRNA-Notch3 cells had a 50% decrease in tumor burden, 50% reduction in osteolytic lesions, and exhibited 30% more cancellous bone compared to mice bearing control MM cells. Together, these preclinical and clinical findings support that Notch3 signaling is a crucial mediator of homotypic and heterotypic communication in the MM tumor niche. Future studies are needed to evaluate Notch3 in the tumor microenvironment as a therapeutic target for the treatment of MM. Citation Format: Hayley M. Sabol, Tania Amorim, Cody Ashby, David Halladay, Judith Anderson, Meloney Cregor, Megan Sweet, Intawat Nookaew, Noriyoshi Kurihara, G. David Roodman, Teresita Bellido, Jesus Delgado-Calle. Notch3 signaling between myeloma cells and osteocytes in the tumor niche promotes tumor growth and bone destruction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5672.