Summary A wealth of physical interaction data between transcription factors (TFs) and DNA has been generated, but these interactions often do not have apparent regulatory consequences. Therefore, equating physical interaction data with gene regulatory networks (GRNs) is problematic. Here, we comprehensively assay TF activity, rather than binding, to construct a network of gene regulatory interactions in the C. elegans intestine. By manually observing the in vivo tissue-specific knockdown of 921 TFs on a panel of fluorescent transcriptional reporters, we identified a GRN of 411 interactions between 19 promoters and 177 TFs. This GRN shows only a modest overlap with physical interactions, indicating that many regulatory interactions are indirect. We applied nested effects modeling to uncover the information flow between TFs in the intestine that converges on a small set of physical TF-promoter interactions. We found numerous cell non-autonomous regulatory interactions, illustrating tissue-to-tissue communication. Our study illuminates the complexity of gene regulation in the context of a living animal.