Disruptor of telomeric silencing 1-like (Dot1l) is a histone 3 lysine 79 methyltransferase. Constitutive Dot1l knockout mice studies show that Dot1l is essential for prenatal development and hematopoiesis. Dot1l also interacts with fusion partners of Mixed Lineage Leukemia (MLL) gene, which is commonly translocated in human leukemia. However, the requirement of Dot1l in postnatal hematopoiesis and leukemogenesis of MLL translocation proteins have not been conclusively shown. With conditional Dot1l knockout mouse model, we examined the consequences of Dot1l loss in postnatal hematopoiesis and MLL translocation leukemia. Deletion of Dot1l led to failure of hematopoietic homeostasis, and Dot1l- deficient cells minimally reconstituted recipient bone marrow. In addition, MLL-AF9 cells required Dot1l for oncogenic transformation, while cells with other leukemic oncogenes such as Hoxa9/Meis1 and E2A- HLF did not. Furthermore, loss of Dot1l affected histone modification at select loci instead of leading to global changes. These findings illustrate a crucial role of Dot1l in normal hematopoiesis, leukemogenesis of specific oncogenes, and histone cross-talk.