Transcription factors (TFs) involved in sexual reproduction in filamentous fungi have been characterized. However, we have little understanding of how these TFs synergize within regulatory networks resulting in sexual development. We investigated 13 TFs in
Fusarium graminearum
, whose knockouts exhibited abortive or arrested phenotypes during sexual development to elucidate the transcriptional regulatory cascade underlying the development of the sexual fruiting bodies. A Bayesian network of the TFs was inferred based on transcriptomic data from key stages of sexual development. We evaluated
in silico
knockout impacts to the networks of the developmental phenotypes among the TFs and guided knockout transcriptomics experiments to properly assess regulatory roles of genes with same developmental phenotypes. Additional transcriptome data were collected for the TF knockouts guided by the stage at which their phenotypes appeared and by the cognate
in silico
prediction. Global TF networks revealed that TFs within the mating-type locus (
MAT
genes) trigger a transcriptional cascade involving TFs that affected early stages of sexual development. Notably,
PNA1
, whose knockout mutants produced exceptionally small protoperithecia, was shown to be an upstream activator for
MAT
genes and several TFs essential for ascospore production. In addition, knockout mutants of
SUB1
produced excessive numbers of protoperithecia, wherein
MAT
genes and pheromone-related genes exhibited dysregulated expression. We conclude that
PNA1
and
SUB1
play central and suppressive roles in initiating sexual reproduction, respectively. This comprehensive investigation contributes to our understanding of the transcriptional framework governing the multicellular body plan during sexual reproduction in
F. graminearum
.
IMPORTANCE
Understanding transcriptional regulation of sexual development is crucial to the elucidation of the complex reproductive biology in
Fusarium graminearum
. We performed gene knockouts on 13 transcription factors (TFs), demonstrating knockout phenotypes affecting distinct stages of sexual development. Using transcriptomic data across stages of sexual development, we inferred a Bayesian network of these TFs that guided experiments to assess the robustness of gene interactions using a systems biology approach. We discovered that the mating-type locus (
MAT
genes) initiates a transcriptional cascade, with
PNA1
identified as an upstream activator essential for early sexual development and ascospore production. Conversely,
SUB1
was found to play a suppressive role, with knockout mutants exhibiting excessive protoperithecia due to abnormally high expression of
MAT
and pheromone-related genes. These findings highlight the central roles of
PNA1
and
SUB1
in regulating other gene activity related to sexual reproduction, contributing to a deeper understanding of the mechanisms of the multiple TFs that regulate sexual development.