The E2a–Pbxl oncoprotein of human pre-B cell leukemia prevents differentiation and maintains continued cell division in cultured myeloid progenitors. Previously, estrogen-dependent forms of E2a–Pbxl were generated that immortalized neutrophil (ECoM-G cells) or monocyte (ECoM-M cells) progenitors and that permitted their terminal differentiation upon estrogen withdrawal. Here, representational difference analysis (RDA) and Affymetrix array analysis are used to identify changes in gene expression that accompany the early differentiation of these cells. The promoters of these genes, whose expression changes upon E2a–Pbxl inactivation, integrate the biochemical mechanism through which E2a–Pbx1 arrests differentiation and maintains cell division. Inactivation of E2a–Pbxl caused the 10- to 80-fold up regulation of a small subset of myeloid differentiation genes (MRP8, Cnlp, NB1, Bactenecin, YM1, Stefin 1, Lipocortin, Lactoferrin, gp91 phox and Ly6-G) and a 10-fold down regulation of the TLE1 corepressor gene, as well as of a group of genes expressed in dividing cells (c-Myc, Nucleophosmin, Spermidine synthase, NOP56, Hnrpa1). Transcription of 97% of cellular genes, including 300 other transcription factor genes (21 Hox genes) and other myeloid genes, varied less than 3-fold, with most varying less than 50%. Therefore, E2a–Pbxl prevents transcription and maintains the cell cycle by a specific rather than a global transcriptional mechanism. Monocyte progenitors were distinguished by persistent expression of IRF8 and of a category of other genes characterized as “interferon-stimulated” (ISG15, ISG20, Ifit1, Ifi202a, Ifi203, Ifi204, Ifi204-related, IRF7 and Ly6-E.1), as well as by the upregulation of the Lrg21 bZip transcription factor gene during late differentiation. The synchronous expression of stage-specific and cell cycle genes regulated by E2a–Pbx1 in these cell lines comprises a model system in which analysis of their promoters can be used as a starting point to backtrack to the transcriptional mechanisms of oncogenesis by E2a–Pbx1.