AbstractSpalt-like transcription factor 1 (SALL1) is a critical regulator of organogenesis and microglia identity. Here we demonstrate that disruption of a conserved microglia-specific super-enhancer interacting with theSall1promoter results in complete and specific loss ofSall1expression in microglia. By determining the genomic binding sites of SALL1 and leveragingSall1enhancer knockout mice, we provide evidence for functional interactions between SALL1 and SMAD4 required for microglia-specific gene expression. SMAD4 binds directly to theSall1super-enhancer and is required forSall1expression, consistent with an evolutionarily conserved requirement of the TGFβ and SMAD homologsDppandMadfor cell-specific expression ofSpaltin theDrosophilawing. Unexpectedly, SALL1 in turn promotes binding and function of SMAD4 at microglia-specific enhancers while simultaneously suppressing binding of SMAD4 to enhancers of genes that become inappropriately activated in enhancer knockout microglia, thereby enforcing microglia-specific functions of the TGFβ–SMAD signaling axis.