Chromosomal translocations that generate oncogenic fusion proteins are causative for most pediatric leukemias and frequently affect the MLL/ KMT2A gene. In vivo modeling of bona fide chromosomal translocations in human hematopoietic stem and progenitor cells is challenging but essential to determine their actual leukemogenic potential. We therefore developed an advanced lentiviral CRISPR-Cas9 vector that efficiently transduced human CD34(+) hematopoietic stem and progenitor cells and induced the t(11; 19)/MLL-ENL translocation. Leveraging this system, we could demonstrate that hematopoietic stem and progenitor cells harboring the translocation showed only a transient clonal growth advantage in vitro. In contrast, t(11; 19)/MLL-ENL-harboring CD34(+) hematopoietic stem and progenitor cells not only showed longterm engraftment in primary immunodeficient recipients, but t(11; 19)/ MLL-ENL also served as a first hit to initiate a monocytic leukemia-like disease. Interestingly, secondary recipients developed acute lymphoblastic leukemia with incomplete penetrance. These findings indicate that environmental cues not only contribute to the disease phenotype, but also to t(11; 19)/ MLL-ENL-mediated oncogenic transformation itself. Thus, by investigating the true chromosomal t(11; 19) rearrangement in its natural genomic context, our study emphasizes the importance of environmental cues for the pathogenesis of pediatric leukemias, opening an avenue for novel treatment options.