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Nearly 20% of patients with myelofibrosis progress to blast phase disease; an aggressive form of acute myeloid leukemia. Although previous studies have implicated loss of TP53or JARID2in progression, by and large the genetic events that lead to conversion to blast phase remain unknown. To identify genes whose loss drives progression, we performed a focused CRISPR/Cas9 screen in which murine Jak2V617Fbone marrow cells expressing Cas9 were transduced with two separate sgRNA libraries of known tumor suppressor genes and subjected to colony replating assays. Transduction of one of the two libraries led to serial replating and enhanced self-renewal of the Jak2V617Fcells. Subsequent DNA sequencing revealed enrichment of all four guides targeting STK11, the gene that encodes LKB1 which regulates a number of key cellular pathways including energy utilization by activation of AMPK. To confirm that loss of Stk11is the event that leads to increased clonogenicity, we collected cells from Jak2V617F/Vav-Cre+and control Vav-Cre+mice and induced Stk11knockout by electroporating Cas9-Stk11sgRNA ribonucleoprotein complexes. Consistent with the screening results, only Jak2V617FVav-Cre+cells with Cas9-Stk11sgRNA showed serial replating. To determine whether Stk11is required for growth of cells with a different driver of enhanced JAK/STAT signaling, we doubly transduced Stk11homozygous floxed bone marrow cells with MPLW515L-mCherry and Cre-GFP to delete Stk11. As expected, cells with both MPLW515Land Cre recombinase showed enhanced self-renewal, while singly infected control cells failed to replate. These results demonstrate that activation of JAK/STAT signaling can overcome the requirement for Stk11in normal hematopoiesis and suggest that STK11loss may be a strong driver of malignant transformation in combination with enhanced JAK-STAT signaling.