Acute myeloid leukemia (AML) is a highly aggressive and challenging form of leukemia, especially in children. Despite advancements in cancer treatment, pediatric AML continues to have a poor prognosis and remains difficult to treat, particularly due to the high risk of relapse and upcoming resistance to conventional therapies. The need for innovative treatment approaches is urgent in order to improve outcomes for these young patients, and cellular immunotherapy is an attractive route to overcome current limitations of classical chemotherapy. However, current targeted (chimeric antigen receptor) cellular immunotherapies are hampered by the lack of specificity for leukemic cells, in particular the leukemia-initiating fraction. In part, this is due to the difficulties in studying this rare cell population, which have been resolved with recent advances in single-cell OMICS. This PhD project aims to characterize the leukemia-initiating population in pediatric AML with flow cytometry-based enrichment combined with cutting-edge single-cell transcriptomics and proteomics as a basis for the development of targeted CAR-T-cell immunotherapies. The project will characterize persistent AML clones during treatment and remission, which can give rise to lethal relapse, identify their cell surface profile, and design and test CAR constructs towards the development of preclinical immunotherapy testing. Noteworthy, innovative patient-derived xenograft models and the integration into the German AML study group emphasize the translational potential of the project. The anticipated outcomes of this research will not only deepen our understanding of the step-wise progression of pediatric AML, but will also advance the development of new therapies capable of eradicating persistent cancer cells, potentially transforming treatment options for AML.