Simple SummaryThe transcription factor Wilms tumor one (WT1) is highly expressed in malignant cells of patients with hematological malignancies, with a considerable percentage of patients harboring deactivating mutations in this gene. To date, the extent of WT1’s contribution to leukemogenesis is still unclear and this, at least in part, results from the complexity of WT1 regulation and is further complicated by the lack of an appropriate model system to studyWT1isoform formation under settings closer to reality. In the present study, we describe a set of leukemia and lymphoma cell lines, which are carefully characterized for WT1’s unique properties and thoroughly screened for sequence alterations inWT1and other genes important for leukemogenesis. We studied the formation of endogenous truncated WT1 and developed a model system for the study of WT1 major isoforms, thus significantly contributing to the field of WT1 research.AbstractWT1is a true chameleon, both acting as an oncogene and tumor suppressor. As its exact role in leukemogenesis is still ambiguous, research with model systems representing natural conditions surrounding the genetic alterations in WT1 is necessary. In a cohort of 59 leukemia/lymphoma cell lines, we showed aberrant expression forWT1mRNA, which does not always translate into protein levels. We also analyzed the expression pattern of the four major WT1 protein isoforms in the cell lines and primary AML blasts with/withoutWT1mutations and demonstrated that the presence of mutations does not influence these patterns. By introduction of key intronic and exonic sequences ofWT1into a lentiviral expression vector, we developed a unique tool that can stably overexpress the fourWT1isoforms at their naturally occurring tissue-dependent ratio. To develop better cellular model systems for WT1, we sequenced large parts of its gene locus and also other important myeloid risk factor genes and revealed previously unknown alterations. Functionally, inhibition of the nonsense-mediated mRNA decay machinery revealed that under natural conditions, the mutated WT1 alleles go through a robust degradation. These results offer new insights and model systems regarding the characteristics of WT1 in leukemia and lymphoma.