The hetR, patA, hetN, and patS genes are part of a regulatory network that regulates the differentiation and patterning of heterocysts in the filamentous cyanobacterium Anabaena sp. strain PCC 7120. In this report, the epistatic interactions of mutant alleles of these four genes have been used to refine our understanding of their relationships to one another. The hetR gene was necessary for differentiation in genetic backgrounds that normally give rise to excessive differentiation, supporting its role as the master regulator of differentiation and indicating that HetR directly regulates factors in addition to hetR and patS genes that regulate differentiation. A functional patS gene was necessary for the delayed multiple-contiguous-heterocyst phenotype observed in hetN mutants as well as for the relative lack of intercalary heterocysts in patA mutants. Epistasis results with mutant alleles of these three genes suggested that PatA attenuates the negative effects of both PatS and HetN on differentiation and promotes differentiation independent of its antagonistic effects on PatS and HetN activity. Cooverxpression of patS and hetR in a synthetic operon indicated that patS acts at a point downstream of hetR transcription in the regulatory network controlling differentiation. A model for the regulation of differentiation that is consistent with these and previous findings is presented.