Fumonisins are carcinogenic mycotoxins produced by the maize ear rot pathogen Gibberella moniliformis (anamorph Fusarium verticillioides). These toxins consist of a linear polyketide-derived backbone substituted at various positions with an amine, one to four hydroxyl, two methyl, and two tricarballylic ester functions. In this study, we generated and characterized deletion mutants of G. moniliformis for five genes, FUM7, FUM10, FUM11, FUM14, and FUM16 in the fumonisin biosynthetic gene cluster. Functional analysis of mutants in four genes, predicted to encode unrelated proteins, affected formation of the tricarballylic esters. FUM7 deletion mutants produced a previously undescribed homologue of fumonisin B1 with an alkene function in both tricarballylic esters, FUM10 and FUM14 deletion mutants produced homologues of fumonisin B3 and fumonisin B4 that lack tricarballylic ester functions, and FUM11 deletion mutants produced fumonisins that lack one of the tricarballylic ester functions. These phenotypes indicated specific roles for FUM7, FUM10, FUM11, and FUM14 in fumonisin biosynthesis that are consistent with the predicted proteins encoded by each gene. Deletion of FUM16 had no apparent effect on fumonisin production. The phenotypes of the deletion mutants provide further insight into the order of steps in fumonisin biosynthesis.