PU.1 is a key transcription factor for hematopoiesis and macrophage differentiation. Using chromatin immunoprecipitation we have previously identified several PU.1 target genes in macrophages and microglia. With the aim to complement these studies, we performed a transcriptomic analysis of PU.1^−/− progenitors after restoration of PU.1 activity. PUER cells committed to macrophage differentiation were analyzed with novel Affymetrix exon 1.0 ST arrays and Affymetrix 430 2.0 genome arrays for crosswise validation. We combined these genome-wide expression data with a publicly-available microarray dataset of PU.1-knockdown hematopoietic stem cells for an integrated analysis. Bibliographic gene connections, binding site prediction and ChIP-Chip data were used to define a multi-level PU.1 regulatory network in macrophages. Moreover, an alternative transcript of the novel PU.1 target gene Ptpro was identified by exon arrays and PU.1 binding to an intronic promoter was demonstrated. In conclusion, we present a PU.1 transcriptional network with novel validated PU.1 target genes.