Eukaryotic DNA synthesis on the lagging strand occurs in a discontinuous fashion via Okazaki fragments. Each fragment is initiated by a short RNA/DNA primer. A processing pathway removes the initiator segment and joins the fragments. Processing is initiated when DNA polymerase delta displaces the RNA/DNA primer into a flap intermediate. The nuclease/helicase Dna2 and flap endonuclease 1 (FEN1) are proposed to cleave these flap intermediates. For cleavage, both enzymes must track from a free 5'-end of the flap toward the site of cleavage. When flaps become long they are bound by the single-stranded binding protein, replication protein A (RPA), which inhibits FEN1 cleavage, but stimulate cleavage by Dna2. After Dna2 cleavage, a short flap, now devoid of RPA, is cleaved by FEN1 to create nick for ligation. These studies focus on understanding the functional interactions among RPA, Dna2, and FEN1 during Okazaki flap processing. We show that Dna2 dissociates flap-bound RPA, independent of cleavage, allowing Dna2 direct access to the flap. In addition, we found that RPA dissociation does not require tracking by Dna2 but needs a genuine flap structure. RPA was also shown to stimulate Dna2 cleavage activity by the removal of DNA secondary structure in the flap. Additionally, we demonstrated that a flap-bound nuclease-defective Dna2 mutant is unable to block FEN1 cleavage on a flap substrate. In fact, FEN1 disengages the tracking mechanism of the flap-bound Dna2. Further studies revealed that FEN 1 disengagement of flap-bound Dna2 prevents blockage of FEN 1 cleavage on short flap intermediates, which Dna2 cannot cleave. These findings suggest that FEN1 dissociates Dna2 to make the flap accessible for FEN1. We further determined that disengagement of Dna2 by FEN1 requires both FEN1 tracking and a genuine flap structure. Finally, the dissociation reactions described were reconstituted using a nuclease-defective Dna2 mutant. Even in the absence of cleavage activity Dna2 overcame RPA inhibition of FEN1 to allow flap cleavage. Taken together, these studies demonstrate a coordinated and ordered reaction between RPA, Dna2, and FEN1, which results in efficient Okazaki flap processing.