Mixed lineage leukemia (MLL) fusion proteins are derived from translocations at 11q23 that occur in aggressive subtypes of leukemia. As a consequence, MLL is joined to different unrelated proteins to form oncogenic transcription factors. Here we demonstrate a direct interaction between several nuclear MLL fusion partners and present evidence for a role of these proteins in histone binding. In two-hybrid studies, ENL interacted with AF4 and AF5q31 as well as with a fragment of AF10. A structure-function analysis revealed that the AF4/AF5q31/AF10 binding domain in ENL coincided with the C-terminus that is essential for transformation by MLL-ENL. The ENL/AF4 association was corroborated by GST-pulldown experiments and by mutual coprecipitation. Both proteins colocalized in vivo in a nuclear speckled pattern. Moreover, AF4 and ENL coeluted on sizing columns together with the known ENL binding partner Polycomb3, suggesting the presence of a multiprotein complex. The overexpression of ENL alone activated a reporter construct and a mutational screen indicated the conserved YEATS domain as essential for this function. Overlay and pulldown-assays finally showed a specific and YEATS domain-dependent association of ENL with histones H3 and H1. In summary, our studies support a common role for nuclear MLL fusion partners in chromatin biology.