Aspergillus flavus is a major virulent fungus of maize and peanuts, which produces aflatoxin B1. A. flavusmainly reproduces through asexual reproduction, producing the asexual spore called conidia. The process of conidia formation (conidiation) is controlled by transcription factors (TFs). In this study, we characterized the role of MYB-like protein A, MylA, in A. flavus. First, we measured the expression of AflmylA during the life cycle of A. flavus and figured out AflmylA was highly expressed in conidia. Then, we assessed the role of MylA in fungal development of A. flavus. The AflmylA deficient mutant showed reduced conidia production and colony growth compared to the control. Also, the AflmylA strain produced reduced amount of sclerotia. Next, we analyzed the role of MylA in A. flavus conidia and found out deletion of AflmylA resulted in reduced trehalose content, spore viability, and stress tolerance in A. flavus. Lastly, we assessed the function of MylA in A. flavus pathogenicity by performing kernel bioassay. When incubated with kernels, the AflmylA null mutant showed reduced conidia colonization and aflatoxin B1 production. Taken together, these results suggest that MylA is essential for proper fungal development, conidial viability, and pathogenicity of A. flavus.