Cytokine-dependent induction of correctly spliced germline (GL) transcripts is required to target the appropriate switch region for class switch recombination. GL transcription is linked to the cell cycle and the number of cell divisions through mechanisms that have not been defined. The human proximal ε GL promoter contains an IL-4 responsive element (IL-4RE) that binds STAT6 and is sufficient to confer IL-4 inducibility to a heterologous promoter in transient transfection studies. We show herein that the IL-4RE contains a novel Myb binding motif that overlaps the 3′ end of the STAT6 palindrome. EMSA analysis showed binding to the IL-4RE of endogenous Myb proteins expressed in BL-2 B cells and Jurkat T cells. However, double occupancy of a probe spanning both STAT6 and Myb binding motifs could not be detected. Thus, binding of either factor may prevent protein/DNA interactions at the other site, raising the possibility that Myb binding may interfere with STAT6-dependent activation of the IL-4RE. Indeed, cotransfection of A-Myb or c-Myb expression vectors in HEK293 and BL-2 cells suppressed STAT6-dependent transcription from a reporter construct containing four copies of the IL-4RE cloned upstream of a minimal thymidine kinase promoter. Most importantly, overexpression of A-Myb was sufficient to suppress IL-4-induced endogenous ε GL transcription in BL-2 cells. Our results indicate that Myb proteins, which are known to act as cell cycle sensors, may play an important mechanistic role in the in vivo regulation of ε GL transcription in human B cells.