The development of mushroom-forming fungi represents a transition from simple to complex multicellularity. Mushroom development starts with localized hyphal branching, giving rise to primary and secondary hyphal knots. However, the gene regulatory networks orchestrating this process remain largely elusive. Here, we identified 37 conserved transcription factors (TFs) in Coprinopsis cinerea, most of them are up-regulated early during fruiting body development among mushroom-forming species. Using CRISPR/Cas9, we generated 37 TFs mutants from the C. cinereaAmutBmut #326 strain. The mutants showed phenotypes ranging from the lack of hyphal knot formation to arrest at the primary/secondary hyphal knot, or later developmental stages. Based on four phenotyping methods, we identified seven TFs that regulate the transitions from vegetative mycelium to primary and secondary hyphal knots. RNA sequencing revealed homogeneity of the gene expression at the vegetative mycelium stage, but divergence during hyphal knot formation between the mutants and the WT strain. Specifically, based on the TFs expression in each mutant and the global transcriptome pattern, we suppose the MatA and the homolog of the sexual/asexual regulator in yeast might function upstream, and the close homolog of Candida albicans morphogenetic TF might function downstream during HK formation. The expression of the other four TFs suggests the potential direct or indirect reciprocal regulation among them, which include the homolog of morphogenetic TF in pathogenetic fungi, and the one which is involved in mushroom formation in Schizophyllum commune. Our study paves the road to identifying key regulators of mushroom formation in Agaricomycetes.