Mapping an organism’s transcriptional regulatory network (TRN), which consists of all interactions between its transcription factors (TF) and target genes, is a necessary step in developing a complete understanding of how that organism normally develops, and how disease states can arise. However, the number of TFs, target genes, and potential regulatory interactions make this a difficult task, even in simple eukaryotes. In this study, the TRN of Schizosaccharomyces pombe was studied. To do so, we systematically deleted over 80% of fission yeast TFs, and characterized the effects of TFΔ on cell growth, length, and gene expression. Deletion of most TFs did not appear to impact the cell, suggesting that many may be inactive in rich medium. To circumvent this issue, we used two approaches. First, hypersensitivity of TFΔ strains to various drug compounds was determined to identify conditions that might induce TF activity. A four-way microarray expression profiling scheme was then used to identify the target genes and function of uncharacterized TF Toe1. This revealed that Toe1 regulated several genes implicated in the pyrimidine salvage pathway. Secondly, we systematically overexpressed S. pombe TF genes under control of the nmt1 promoter. Over 70% of the overexpressed TF genes resulted in altered cell length or fitness, indicating that their target genes might be inappropriately expressed. Expression microarrays and ChIP-chip were thus used to identify the putative target genes for three uncharacterized fungal TFs, SPBC1773.16, SPBC16G5.17, and SPAC25B8.11, revealing potential roles for each of these TFs in regulating the utilization of alternative nitrogen sources. Finally, screens of our TFΔ array in flocculation inducing and rich mediums revealed six novel transcriptional activators (Foe1, Prr1, Prt1, SPBC530.08, Fep1, and Grt1) and two transcriptional repressors (Scr1 and SPBC56F2.05) of flocculation. Microarray expression profiling was used to identify potential target genes for six of these TFs. Additionally, ChIP-chip was used to identify direct Foe1 targets, revealing that this TF directly binds and regulates the expression of several genes encoding flocculins and cell wall remodeling/biosynthesis proteins. Collectively, these results should contribute to a better understanding of transcriptional regulation in S. pombe as a whole.