Developmental transcription factors act in networks, but how these networks achieve cell- and tissue specificity is still poorly understood. Here, we explored pre-B cell leukemia homeobox 1 (PBX1) in adult neurogenesis combining genomic, transcriptomic, and proteomic approaches. ChIP-seq analysis uncovered PBX1 binding to numerous genomic sites. Integration of PBX1 ChIP-seq with ATAC-seq data predicted interaction partners, which were subsequently validated by mass spectrometry. Whole transcriptome spatial RNA analysis revealed shared expression dynamics of Pbx1 and interacting factors. Among these were class I bHLH proteins TCF3 and TCF4. RNA-seq following Pbx1, Tcf3 or Tcf4 knockdown identified proliferation- and differentiation associated genes as shared targets, while sphere formation assays following knockdown argued for functional cooperativity of PBX1 and TCF3 in progenitor cell proliferation. Notably, while physiological PBX1-TCF interaction has not yet been described, chromosomal translocation resulting in genomic TCF3::PBX1 fusion characterizes a subtype of acute lymphoblastic leukemia. Introducing Pbx1 into Nalm6 cells, a pre-B cell line expressing TCF3 but lacking PBX1, upregulated the leukemogenic genes BLK and NOTCH3, arguing that functional PBX1-TCF cooperativity likely extends to hematopoiesis. Our study hence uncovers a transcriptional module orchestrating the balance between progenitor cell proliferation and differentiation in adult neurogenesis with potential implications for leukemia etiology.