CCAAT enhancer binding protein epsilon (C/EBPepsilon) is a myeloid specific transcription factor that is essential for terminal granulocytic differentiation. Retinoblastoma (Rb) and E2F1 are critical cell cycle regulators that also have been implicated in several differentiation systems. Here, we demonstrate that C/EBPepsilon interacts with Rb and E2F1 during granulocytic differentiation in NB4 and U937 human myeloid cells and in 32Dcl3 murine myeloid precursor cells. The interaction between C/EBPepsilon and Rb enhances C/EBPepsilon-mediated transcription of myeloid specific genes both in reporter assays and endogenously. The C/EBPepsilon-E2F1 interaction results in repression of E2F1-mediated transcriptional activity. Finally, overexpression of C/EBPepsilon in human myeloid cells leads to down-regulation of c-Myc. We propose that the interactions between C/EBPepsilon, a tissue-specific transcription factor, and the broad-spectrum proteins, Rb and E2F1, are important in C/EBPepsilon-induced terminal granulocytic differentiation.