The radical S-adenosyl-L-methionine (SAM) superfamily is a large and growing group of enzymes that conduct complex radical-mediated transformations. A one-electron reduction of SAM via the +1 state of the cubane [4Fe-4S] cluster generates a 5'-deoxyadenosyl radical, which initiates turnover. The [4Fe-4S] cluster must be reduced from its resting +2 state to the catalytically active +1 oxidation state by an electron. In practice, dithionite or the Escherichia coli flavodoxin (EcFldA)/ferredoxin (flavodoxin):NADP(+) oxidoreductase (Fpr)/NADPH system is used. Herein, we present a systematic investigation of the reductive activation of the radical SAM enzyme CDG synthase (BsQueE) from Bacillus subtilis comparing biological and chemical reductants. These data show that either of the flavodoxin homologues encoded by the B. subtilis genome, BsYkuN or BsYkuP, as well as a series of small molecule redox mediators, supports BsQueE activity. With dithionite as a reductant, the activity of BsQueE is ~75-fold greater in the presence of BsYkuN and BsYkuP compared to that in the presence of dithionite alone. By contrast, EcFldA supports turnover to ~10-fold greater levels than dithionite alone under the same conditions. Comparing the ratio of the rate of turnover to the apparent binding constant for the flavodoxin homologues reveals 10- and 240-fold preferences for BsYkuN over BsYkuP and EcFldA, respectively. The differential activation of the enzyme cannot be explained by the abortive cleavage of SAM. We conclude from these observations that the differential activation of BsQueE by Fld homologues may reside in the details of the interaction between the flavodoxin and the radical SAM enzyme.