The molecular pathways of normal myeloid differentiation, as well as the mechanisms by which oncogenes disrupt this process, remain poorly understood. A major limitation in approaching this problem has been the lack of suitable cell lines that exhibit normal, terminal, and synchronous differentiation in the absence of endogenous oncoproteins and in response to physiologic cytokines, and whose differentiation can be arrested by ectopically expressed human oncoproteins. This report describes clonal, granulocyte-macrophage colony-stimulating factor-dependent myeloid cell lines that exhibit these properties. The cell lines were established by conditional immortalization of primary murine marrow progenitors with an estrogen-regulated E2a/Pbx1-estrogen receptor fusion protein. Clones were identified that proliferated as immortalized blasts in the presence of estrogen, and that exhibited granulocytic, monocytic, or bipotential (granulocytic and monocytic) differentiation on estrogen withdrawal. Differentiation was normal and terminal as evidenced by morphology, cell surface markers, gene expression, and functional assays. The differentiation of the cells could be arrested by heterologous oncoproteins including AML1/ETO, PML/RARalpha, PLZF/RARalpha, Nup98/HoxA9, and other Hox proteins. Furthermore, the study examined the effects of cooperating oncoproteins such as Ras or Bcr/Abl, which allowed for both factor-independent proliferation and differentiation, or Bcl-2, which permitted factor-independent survival but not proliferation. These myeloid cell lines provide tools for examining the biochemical and genetic pathways that accompany normal differentiation as well as a system in which to dissect how other leukemic oncoproteins interfere with these pathways.