[FeFe] hydrogenases catalyze H(2) production using the H-cluster, an iron-sulfur cofactor that contains carbon monoxide (CO), cyanide (CN(-)), and a dithiolate bridging ligand. The HydE, HydF, and HydG maturases assist in assembling the H-cluster and maturing hydrogenases into their catalytically active form. Characterization of these maturases and in vitro hydrogenase activation methods have helped elucidate steps in the H-cluster biosynthetic pathway such as the HydG-catalyzed generation of the CO and CN(-) ligands from free tyrosine. We have refined our cell-free approach for H-cluster synthesis and hydrogenase maturation by using separately expressed and purified HydE, HydF, and HydG. In this report, we illustrate how substrates and protein constituents influence hydrogenase activation, and for the first time, we show that each maturase can function catalytically during the maturation process. With precise control over the biomolecular components, we also provide evidence for H-cluster synthesis in the absence of either HydE or HydF, and we further show that hydrogenase activation can occur without exogenous tyrosine. Given these findings, we suggest a new reaction sequence for the [FeFe] hydrogenase maturation pathway. In our model, HydG independently synthesizes an iron-based compound with CO and CN(-) ligands that is a precursor to the H-cluster [2Fe](H) subunit, and which we have termed HydG-co. We further propose that HydF is a transferase that stabilizes HydG-co and also shuttles the complete [2Fe](H) subcluster to the hydrogenase, a translocation process that may be catalyzed by HydE. In summary, this report describes the first example of reconstructing the [FeFe] hydrogenase maturation pathway using purified maturases and subsequently utilizing this in vitro system to better understand the roles of HydE, HydF, and HydG.