We have developed a high-throughput method for creating Neurospora knockout mutants.  In this procedure, yeast recombinational cloning is used to create constructs that are then transformed into Neurospora strains deficient in nonhomologous end-joining DNA repair (mus-51 and mus-52 mutants).  Here we present the results of our initial application of the procedure, with mutational analysis of 103 transcription factor-encoding genes.  The methodology is robust, with a >90% success rate for producing the desired knockout mutant.  The resulting mutants were screened for a variety of phenotypes and 43% exhibited discernible defects.  The genes producing phenotypes are variously involved in growth of basal hyphae (25 genes), aerial hyphae height and/or macroconidiation (27 genes), and differentiation of protoperithecia or perithecia (15 genes).  This analysis demonstrated roles for many uncharacterized transcription factors and also revealed novel functions for genes that had been previously studied.  Several transcription factors are required for than one aspect of growth or development, suggesting roles in integration of multiple upstream signals.  The observation that half of the genes did not produce obvious defects when mutated may result from functional redundancy, which has been reported for other transcription factor genes.  The availability of this collection of Neurospora transcription factor mutants will enable future investigations aimed at elucidating the complexity of gene regulation in filamentous fungi.