To identify developmentally regulated genes during myeloid differentiation, a self‐inactivating retroviral gene‐trap vector carrying a β‐galactosidase‐neomycin (SA/lacZ/neo) fusion gene was constructed and used to infect myeloid progenitor cells (FDCP‐Mix A4). G418‐resistant and β‐galactosidase positive cell lines (gene‐trap integration [GTI] clones) were established and induced to differentiate in vitro into either macrophages or granulocytes. Expression of the trapped loci was monitored at a single‐cell level by analysing the mature cell types for β‐galactosidase activity. All 37 GTI clones tested showed down‐regulation either during granulocyte or both granulocytic and macrophage differentiation. The endogenous coding regions fused to the SA/lacZ/neo reporter gene were isolated from eight clones. Molecular analysis revealed that half of them represented novel mouse genes (def‐2, ‐3, ‐6 and ‐8) which we confirmed to be differentially expressed in primary haemopoietic tissues. Database searches revealed no significant similarities for def‐2 (associated with haemopoietic progenitors) and def‐8 (expressed most strongly in peripheral leucocytes). Def‐6, which is down‐regulated upon the differentiation into myeloid as well as erythroid lineages, was found to be closely related but not identical with the recently described B‐cell‐specific switch recombinase SWAP‐70. Def‐3, which is down‐regulated upon differentiation into granulocytes but expressed in progenitor cells and macrophages, defines a novel family of RNA binding proteins.