Hematopoiesis consists of a series of lineage decisions controlled by specific gene expression that is regulated by transcription factors and intracellular signaling events in response to environmental cues. Here, we demonstrate that the balance between Eprotein transcription factors and their inhibitors, Id proteins, is important for the myeloidversus-lymphoid fate choice. Using Id1-GFP knockin mice, we show that transcription of the Id1 gene begins to be up-regulated at the granulocyte-macrophage progenitor stage and continues throughout myelopoiesis. Id1 expression is also stimulated by cytokines favoring myeloid differentiation. Forced expression of Id1 in multipotent progenitors promotes myeloid development and suppresses B-cell formation. Conversely, enhancing E-protein activity by expressing a variant of E47 resistant to Id-mediated inhibition prevents the myeloid cell fate while driving B-cell differentiation from lymphoid-primed multipotent progenitors. Together, these results suggest a crucial function forEproteins in the myeloid-versuslymphoid lineage decision. © 2009 by The American Society of Hematology.