The pseudokinase MLKL is the terminal effector of necroptosis, a caspase-independent pro-inflammatory programmed cell death pathway. MLKL interaction with, and phosphorylation by protein kinase RIPK3 is required for the killing activity of MLKL, but the precise mechanism governing their interaction and activation is incompletely understood. To examine the mechanism of human MLKL-RIPK3 interactions and activations, we characterized Monobodies (Mb), synthetic binding proteins, that bind the pseudokinase domain of MLKL within human cells and their crystal structures in complex with the human MLKL pseudokinase domain, as well as a recombinant human RIPK3:MLKL complex. Mb32 binds MLKL hinge region and co-crystallized with MLKL in a novel open conformation. Mb27, whose epitope partially overlaps with RIPK3 interface, binds MLKL only after necroptotic stimulation in cells, and bind mutually exclusively from RIPK3 in recombinant RIPK3:MLKL complex. Recombinant RIPK3 and MLKL form a stable complex when overexpressed in insect cells, but the purified protein complex dissociates upon Mg2+/ATP treatment. Together, these studies provide further evidence that MLKL undergoes a large conformational change upon activation and suggest MLKL disengagement from RIPK3 as a potential checkpoint in the necroptosis pathway.