Iron-sulfur clusters are key iron cofactors in biological pathways ranging from nitrogen fixation to respiration. Due to the toxicity of ferrous iron and sulfide to the cell, in vivo Fe-S cluster assembly is carried out by multi-protein biosynthetic pathways. Fe-S cluster assembly proteins traffic iron and sulfide, assemble nascent Fe-S clusters, and correctly transfer Fe-S clusters to the appropriate target metalloproteins in vivo. The gram-negative bacterium E. coli contains a stress-responsive Fe-S cluster assembly system, the SufABCDSE pathway, that functions under iron starvation and oxidative stress conditions that compromise Fe-S homeostasis. Using a combination of protein-protein interaction and in vitro Fe-S cluster assembly assays, we have characterized the relative roles of the SufBCD complex and the SufA protein during Suf Fe-S cluster biosynthesis. These studies reveal that SufA interacts with SufBCD in order to accept Fe-S clusters formed de novo on the SufBCD complex. Our results represent the first biochemical evidence that the SufBCD complex within the Suf pathway functions as a novel Fe-S scaffold system to assemble nascent clusters and transfer them to the SufA Fe-S shuttle.