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FACT (facilitates chromatin transcription) is a histone chaperone that is involved in the processes of transcription initiation and elongation, DNA replication and repair [1]. Yeast FACT conducts large-scale ATP-independent nucleosome unfolding [2]; the mechanism of this process is unknown. Here we determined the role of Nhp6 protein and C-terminal domains of yeast FACT complex in nucleosome unfolding using single-particle Förster resonance energy transfer (spFRET) microscopy, EMSA and FRET-in-gel methods. Nhp6 protein interacts with the C-terminal domains of Spt16 and Pob3 subunits of yFACT, inducing unfolding of FACT in the absence of nucleosomes. Analysis of yeast FACT interaction with nucleosomes and hexasomes suggests that Spt16 subunit of FACT drives nucleosome unfolding, while Pob3 subunit is required for complete unfolding. Thus, FACT-dependent nucleosome unfolding is a tightly coordinated process that requires Nhp6 protein and C-terminal domains of Spt16 and Pob3 subunits of yFACT. Our data suggest a detailed model of FACT-dependent nucleosome unfolding.