PU.1 is essential for the differentiation of haemopoietic precursors and is strongly implicated in leukaemogenesis, yet the protein interactions that regulate its activity in different myeloid lineages are still largely unknown. In the present study, by combining fluorescent EMSA (electrophoretic mobility-shift assay) with MS, we reveal the presence of hnRNP K (heterogeneous nuclear ribonucleoprotein K) in molecular complexes that PU.1 forms on the CD11b promoter during the agonist-induced maturation of AML (acute myeloid leukaemia)-derived cells along both the granulocytic and the monocytic lineages. Although hnRNP K and PU.1 act synergistically during granulocytic differentiation, hnRNP K seems to have a negative effect on PU.1 activity during monocytic maturation. Since hnRNP K acts as a docking platform, integrating signal transduction pathways to nucleic acid-directed processes, it may assist PU.1 in activating or repressing transcription by recruiting lineage-specific components of the transcription machinery. It is therefore possible that hnRNP K plays a key role in the mechanisms underlying the specific targeting of protein-protein interactions identified as mediators of transcriptional activation or repression and may be responsible for the block of haemopoietic differentiation.