Discovering the genes underlying fundamental processes that enable cells to live and reproduce is a technical challenge, because loss of gene function in mutants results in organisms that cannot survive. This study describes a forward genetics method to identify essential genes in fungi, based on the propensity for Agrobacterium tumefaciens to insert T-DNA molecules into the promoters or 5' untranslated regions of genes and by placing a conditional promoter within the T-DNA. Insertions of the promoter of the GAL7 gene were made in the human pathogen Cryptococcus neoformans. Nine strains of 960 T-DNA insertional mutants screened grew on media containing galactose, but had impaired growth on media containing glucose, which suppresses expression from GAL7. T-DNA insertions were found in the homologs of IDI1, MRPL37, NOC3, NOP56, PRE3 and RPL17, all of which are essential in ascomycete yeasts Saccharomyces cerevisiae or Schizosaccharomyces pombe. Altering the carbon source in the medium provided a system to identify phenotypes in response to stress agents. The pre3 proteasome subunit mutant was further characterized. The T-DNA insertion and phenotype co-segregate in progeny from a cross, and the growth defect is complemented by the reintroduction of the wild type gene into the insertional mutant. A deletion allele was generated in a diploid strain, this heterozygous strain was sporulated, and analysis of the progeny provided additional genetic evidence that PRE3 is essential. The experimental design is applicable to other fungi and has other forward genetic applications such as to isolate over-expression suppressors or enhance the production of traits of interest.