The production of terminally differentiated granulocytes and monocytes occurs by a program of orderly and sequential gene expression. This genetic program can be studied in vitro utilizing granulocyte-monocyte colony stimulating factor (GM-CSF) to stimulate proliferation and myeloid cell maturation. We have identified a series of genes expressed during early myeloid differentiation by differential screening of a cDNA library prepared from GM-CSF stimulated murine progenitor cells. From 72 potential early myeloid specific clones, three (B9, C9, C15) were characterized further. The time course of RNA accumulation during GM-CSF stimulated maturation demonstrated a unique pattern for each clone. B9 was expressed predominantly on day 3, and followed a pattern of accumulation similar to that for myeloperoxidase. (Jaffe et al. (1988), Oncogene, 2, 167-174). C9 expression increased gradually to a maximum level on day 3 and then plateaued; it appeared to be equally expressed in granulocytes and monocytes. C15 was expressed at a consistently high level in unstimulated cells and at 1-3 days post GM-CSF stimulation. It then decreased to undetectable levels. Hybridization of these clones to a panel of murine tissue RNAs demonstrated restricted expression of B9 to bone marrow, while C9 was present in most murine tissues, including thymus. C15 expression was relatively restricted to ovary/uterus, liver and adrenal, in addition to bone marrow. Partial DNA sequence analysis suggested that B9 was a novel sequence not previously identified. C9 was identified as thymosin beta-4, and C15 showed extensive homology to lactotransferrin. Thus, screening a bone marrow cDNA library by differential hybridization has successfully yielded a series of DNA sequences regulated during murine myelopoiesis. These include novel sequences (B9), genes previously known to be regulated during myelopoiesis (C15), as well as sequences not recognized previously as being associated with myeloid differentiation (C9).