It is known that transcription factors (TFs) work in cooperation with each other to govern gene expression and thus single TF studies may not always reflect the underlying biology. Using microarray data obtained from two independent studies of the first wave of spermatogenesis, we tested the hypothesis that co-expressed spermatogenic genes in cells committed to differentiation are regulated by a set of distinct combinations of TF modules. A computational approach was designed to identify over-represented module combinations in the promoter regions of genes associated with transcripts that either increase or decrease in abundance between the first two major spermatogenic cell types: spermatogonia and spermatocytes. We identified five TFs constituting four module combinations that were correlated with expression and repression of similarly regulated genes. These modules were biologically assessed in the context that they represent the key transcriptional mediators in the developmental transition from the spermatogonia to spermatocyte.