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Acute myeloid leukemia (AML) is a complex, heterogeneous disease with variable outcomes following curative intent chemotherapy. AML with inv(3) is a genetic subgroup characterized by a very low response rate to current induction type chemotherapy and thus has among the worst long-term survivorship of the AMLs. Here, we describe OCI-AML-20, a new AML cell line with inv(3) and deletion of chromosome 7; the latter is a common co-occurrence in inv(3) AML. In OCI-AML-20, CD34 expression is maintained and required for repopulation in vitro and in vivo. CD34 expression in OCI-AML-20 shows dependence on the co-culture with stromal cells. Transcriptome analysis indicates that the OCI-AML-20 clusters with other AML patient data sets that have poor prognosis, as well as other AML cell lines, including another inv(3) line, MUTZ-3. OCI-AML-20 is a new cell line resource for studying the biology of inv(3) AML that can be used to identify potential therapies for this poor outcome disease.