We identified and attempted to disrupt the Cryptococcus neoformans homoserine and/or threonine biosynthetic genes encoding aspartate kinase (HOM3), homoserine kinase (THR1) and threonine synthase (THR4); however, each gene proved recalcitrant to disruption. By replacing the endogenous promoters of HOM3 and THR1 with the copper-repressible CTR4-1 promoter, we showed that HOM3 and THR1 were essential for the growth of C. neoformans in rich media, when ammonium was the nitrogen source, or when threonine was supplied as an amino acid instead of a dipeptide. Moreover, the severity of the growth defect associated with HOM3 or THR1 repression increased with increasing incubation temperature. We believe this to be the first demonstration of threonine biosynthetic genes being essential in a fungus. The necessity of these genes for C. neoformans growth, particularly at physiologically relevant temperatures, makes threonine biosynthetic genes ideal anti-cryptococcal drug targets.