Abstract The aggressive and unpredictable behavior of relapsed T-cell acute lymphoblastic leukemia (T-ALL) presents a major clinical challenge, with >70% of children and >90% of adults unable to survive relapsed disease. Relapsed T-ALL often acquires mutations that are not found in the primary malignancy. It is these new mutations that allow clones to survive treatment and drive relapse growth. In order to identify the genes and pathways responsible for T-ALL relapse, we have developed a transgenic zebrafish model of relapsed T-ALL where single fluorescently-labeled leukemic cells are transplanted into genetically identical recipient fish and functionally assessed for differences in relapse growth. Using serial transplantation of single T-ALL cells and >6,000 recipient animals, we have followed single-cell evolution of T-ALL and identified critical drivers of relapse. These experiments showed that 6 of 49 individual T-ALL cells significantly increased their ability to form relapse over time. Analysis of T-ALL clones pre- and post-evolution showed that AKT pathway activation was correlated with increased relapse potential. Subsequent studies utilizing transgenic zebrafish that over-expressed activated AKT in developing T-ALL demonstrated that AKT signaling increased relapse potential 10-fold. Transgenic epistatic experiments revealed that AKT signaling plays two distinct roles in T-ALL relapse: the AKT/mTORC1 pathway directly enhanced relapse potential, while AKT mediated stabilization of the Myc protein increased T-ALL aggressiveness. Moreover, small molecule inhibition of AKT signaling reduced T-ALL relapse potential in vivo by 25-fold and synergized with Dexamethasone, a common cytotoxic chemotherapy, to significantly enhance cell killing in both zebrafish and human T-ALL. Activation of AKT signaling is associated with poor prognosis and drug resistance in human T-ALL, which, together with our work, suggests that AKT will be a useful molecular target in the treatment of T-ALL. Our experiments have documented the functional heterogeneity of single leukemic cells and identified AKT as a critical driver of T-ALL aggression, relapse formation, and insensitivity to therapy. These are first studies performed in any model to follow single cell evolution as it relates to relapse, opening new and exciting avenues of study to uncover genetic pathways that drive cancer malignancy. Citation Format: Jessica S. Blackburn, Sali Liu, Kimberly Dobrinski, Jayme Ranalli, Sarah Martinez, Charles Lee, David Langenau. Single cell evolution of AKT pathway activation drives T-cell acute lymphoblastic leukemia relapse. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3805. doi:10.1158/1538-7445.AM2013-3805