ABSTRACTNF90 and splice variant NF110 are DNA‐ and RNA-binding proteins encoded by the Interleukin enhancer-binding factor 3 (ILF3) gene that regulate RNA splicing, stabilization and export. The role of NF90 in regulating transcription as a DNA-binding protein has not been comprehensively characterized. Here, ENCODE ChIP-seq identified 9,081 genomic sites specifically bound by NF90/110 in K562 cells. One third of binding sites occurred at promoters of annotated genes. NF90/110 binding colocalized with chromatin marks associated with active promoters and strong enhancers. Comparison with 150 ENCODE ChIP-seq experiments revealed that NF90 clustered with transcription factors exhibiting preference for promoters over enhancers (POLR2A,MYC,YY1). Differential gene expression analysis following shRNA knockdown of NF90 in K562 cells revealed that NF90 directly activates transcription factors that drive growth and proliferation (EGR1,MYC), while attenuating differentiation along erythroid lineage (KLF1). NF90/110 binds chromatin to hierarchically regulate transcription factors to promote proliferation and suppress differentiation.