Acute Myeloid Leukemia (AML) in adults is a clinically devastating disease with a 5-year survival rate of only 25%. We lack new and effective therapies for AML, and the chemotherapy standard of care remains unchanged in thirty years. One success story has been the discovery of drugs that trigger the differentiation of leukemic blasts in a small subset of patients, approximately 10%, with acute pro-myelocytic leukemia. Differentiation therapy is not available for the remaining 90% of acute myeloid leukemia patients. We generated a novel cell line model of acute myeloid leukemia, based on the potent ability of the homeobox protein HoxA9 to block differentiation in primary murine cultures of immature myeloblasts. These cells were engineered with a built-in reporter of differentiation, permitting a high-throughput flow-cytometry-based phenotypic differentiation screen against more than 330,000 small molecules. 2,500 compounds were retested resulting in twenty-nine confirmed hits. Active compounds and analogs were obtained, validated, and tested in dose against the screening cell line as well as other murine and human cell lines. This identified twelve active compounds from which two distinct scaffolds were chosen for further study based upon chemical tractability and availability. One of the two hits demonstrated stereospecificity, hence we chose to explore SAR about this lead. Of approximately thirty synthesized analogs, we identified four compounds that were more active in three cell lines (THP-1, U937 and ERHOXA9). We have chosen compound ML390 as the probe given its potent activity. Of note, the lead compound and its analog ML390 are both very well-tolerated with limited cytotoxicity at high concentrations when assayed against cultures of normal human primary bone marrow cells. ML390 exerts its potent differentiation effect on multiple leukemia models, though its mechanism of action is currently unknown. Target identification assays as well as experiments for testing the compound in vivo are currently underway. Mechanism of action studies will be performed in parallel using a combination of gene expression studies (Library of Integrated Network-Based Cellular Signatures, LINCS), stable isotope labeling by amino acids in cell culture (SILAC), and next-generation sequencing of compound-resistant cell clones. ML390 will be tested in mice harboring a HoxA9-driven acute myeloid leukemia to assess its differentiation effect as well as its effect on leukemic progression and overall survival. The chemotherapy standard of care in acute myeloid leukemia relies on traditional cytotoxic chemotherapy and has not changed in more than thirty years. We need new, and less toxic, therapeutic agents. In the context of our novel cell line model of AML, we hope to have identified a clinically relevant pro-differentiation therapy for acute myeloid leukemia. If successful, we anticipate that ML390 will offer insight into the mechanism of overcoming differentiation arrest, and will translate into a starting point for a much-needed new and potent treatment for patients with acute myeloid leukemia.