The Ikaros gene encodes a zinc finger transcription factor that is selectively expressed by all hematopoietic cells. Although Ikaros is required for lymphocyte differentiation, its role in the myeloid lineage is unclear. We show here that Ikaros expression is temporally regulated during neutrophil differentiation: Ikaros is primarily expressed at immature stages and significantly less so in mature neutrophils. Furthermore Ik^L/L mice, harboring a hypomorphic mutation at the Ikaros locus, exhibit several defects during neutrophil differentiation. (1) Ik^L/L fetal livers contain high numbers of neutrophil lineage cells, and this increase is reflected in the number of GM-CSF-dependent progenitor cells. (2) The migratory potential and survival of neutrophil progenitors is altered in vitro. (3) Expression of the Gr-1 marker is delayed and repressed. In contrast, neutrophil function appears normal. These data demonstrate that Ikaros regulates early neutrophil differentiation but is dispensable in mature neutrophils.