Iron deficiency chlorosis (IDC) is a form of abiotic stress that negatively impacts soybean yield. In a previous study, we demonstrated that the historical IDC quantitative trait locus (QTL) on soybean chromosome Gm03 was composed of four distinct linkage blocks, each containing candidate genes for IDC tolerance. Here, we take advantage of virus-induced gene silencing (VIGS) to validate the function of three high-priority candidate genes, each corresponding to a different linkage block in the Gm03 IDC QTL. We built three single-gene constructs to target (, Glyma.03G128300), (, Glyma.03G130000), and (, Glyma.03G130400 and Glyma.03G130600). Given the polygenic nature of the iron stress tolerance trait, we also silenced the genes in combination. We built two constructs targeting + and +. All constructs were tested on the iron-efficient soybean genotype Clark grown in iron-sufficient conditions. We observed significant decreases in soil plant analysis development (SPAD) measurements using the construct and both double constructs, with potential additive effects in the + construct. Whole genome expression analyses (RNA-seq) revealed a wide range of affected processes including known iron stress responses, defense and hormone signaling, photosynthesis, and cell wall structure. These findings highlight the importance of GmGLU1 in soybean iron stress responses and provide evidence that IDC is truly a polygenic trait, with multiple genes within the QTL contributing to IDC tolerance. Finally, we conducted BLAST analyses to demonstrate that the Gm03 IDC QTL is syntenic across a broad range of plant species.