The immunosuppressant Interleukin-10 (IL-10) was identified bioinformatically as a candidate downstream target of the Wilms' tumor transcription factor WT1. As the co-expression of WT1 and IL-10 in monocytic cells suggested biological relevance of this interaction, we investigated the relationship of both factors in vitro and in vivo. Silencing of Wt1 by siRNA reduced IL-10 transcript levels by approximately 90%. IL-10 transcripts were increased more than 15-fold in upon forced expression of Wt1 in transient co-transfection assays as well as in UB27 cells stably transfected with the transcriptionally active Wt1(-KTS) isoform. Interestingly, the Wt1(+KTS) protein, which had previously been associated mainly with posttranscriptional regulation, could be shown to be as efficient in stimulating the IL-10 promoter as the Wt1(-KTS) isoform. Chromatin immunoprecipitation as well as electrophoretic mobility shift assays revealed a cis-element responsible for activation of the IL-10 promoter by Wt1 in murine macrophagocytic cells in vitro and in vivo. Site-directed mutagenesis of the Wt1 binding motif abrogated stimulation of the IL-10 promoter by tumor necrosis factor-a (TNFa). Moreover, when we compared IL-10 mRNA expression in embryonic wildtype and Wt1^-/- mice, we detected significantly decreased IL-10 levels in the livers of Wt1-deficient mice at day E11.5. These results suggest a novel immune regulatory function of Wt1 exerted through a transcriptional control of the IL-10 gene.