Abstract BRAF is the most common driver oncogene in cutaneous melanoma. Activating mutations in the BRAF gene are enriched in melanomas associated with intermittent sun exposure, with the most frequent mutations occurring at codon 600, leading to a constitutive activation of the RAF/MEK/ERK signaling pathway. In previous studies, we demonstrated that ultraviolet radiation (UVR) accelerates melanomagenesis in a mouse model of melanoma driven by BRAFV600E. From whole exome sequencing analysis of the tumors we found that Map3k1 was the most recurrently mutated gene across different experimental cohorts. MAP3K1 is unique in having both a kinase and E3 ligase function, allowing it to regulate protein phosphorylation and ubiquitin-mediated proteasome degradation. The vast majority of the mutations in Map3k1 occurred within the RING domain, which contains the E2 binding site for ubiquitin-conjugating enzymes. The most prevalent non-synonymous mutations where present in codon 484 and arose as a consequence of C-to-T transitions at a dipyrimidine site, characteristic of UVR-induced DNA damage. To understand better the role of MAP3K1, we generated Tyr::CreERT2+/o;Map3k1f/f mice allowing us to conditionally delete Map3k1 specifically in the melanocytes upon topical application of tamoxifen to the dorsal skin of juvenile mice. These mice did not develop tumors, but when crossed to mice carrying a Braf+/LSL-V600E allele, all mice develop melanoma, with a shorter latency and an increased multiplicity compared to Tyr::CreERT2+/o;Braf+/LSL-V600E mice. Tumors showed similar histopathological features, but melanomas carrying Map3k1 floxed or mutant variants presented higher levels of phosphorylated ERK1/2 than those with wild-type Map3k1. In addition, analysis of expression data of human cutaneous melanomas from The Cancer Genome Atlas showed an inverse correlation between the expression of MAP3K1 and ERK2 protein levels. Taken together, our results suggest that MAP3K1 loss-of-function cooperates with BRAFV600E to drive melanoma development by increasing MEK-ERK pathway activation. Citation Format: Lucas D. Trucco, Piyushkumar A. Mundra, Pablo Garcia-Martinez, Kate Hogan, Nathalie Dhomen, Valeria Pavet, Richard Marais. Melanocyte specific deletion of Map3k1 reveals its role in BRAFV600E-driven melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4653.