Multiple myeloma (MM) is an incurable malignancy characterised by uncontrolled proliferation of plasma cells (PCs) in the bone marrow (BM). The development of MM tumours is not only dependent on the cancer cell’s oncogenic mutations but also on selective pressures applied by the BM microenvironment. Hence, we hypothesised that identifying genes whose expression promotes MM cell growth and survival in the BM tumour microenvironment (TME) will facilitate the identification of new druggable targets. To identify novel gene dependencies involved in MM tumour survival in the TME, we used a functional screen combining CRISPR-Cas9 gene editing, using a single-guide RNA (sgRNA) library targeting thousands of genes, with a murine model of MM. The Bassik laboratory apoptosis and cancer CRISPR knockout library was used to transduce the human MM cell-line OPM2 with 31,324 unique sgRNAs targeting 3,015 genes. The modified cell-line (OPM2-Cas9-sgRNA) was injected into the tibiae of immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice and four weeks post-injection, primary tumours were isolated. sgRNA frequencies were assessed via next generation sequencing and compared with the initial library and the injected cells using the MAGeCK algorithm. We identified 28 genes where CRISPR knockout had no significant effect in vitro but were critical dependencies for tumour development, with sgRNAs undetectable (p<0.05) in vivo. A further 115 genes were significantly depleted (p<0.05) in vitro and were undetectable in vivo, suggesting involvement in both in vitro cell proliferation and tumour formation. Gene set enrichment analysis identified genes essential for in vitro survival and growth were predominantly involved in cell survival pathways; while, genes that were required uniquely for survival and growth in vivo were predominantly involved in cell metabolism pathways. This study has successfully identified novel genes required for in vivo MM tumour development. These genes are currently being validated using a secondary genetic screen and will be further investigated using patient derived data sets and multiple MM cell-lines to investigate their potential as therapeutic targets.