Members of the SCY1-like (SCYL) family of protein kinases are evolutionarily conserved and ubiquitously expressed proteins characterized by an N-terminal pseudokinase domain, centrally located Huntingtin, elongation factor 3, protein phosphatase 2A, yeast kinase TOR1 repeats, and an overall disorganized C-terminal segment. In mammals, three family members encoded by genesScyl1,Scyl2, andScyl3have been described. Studies have pointed to a role for SCYL1 and SCYL2 in regulating neuronal function and viability in mice and humans, but little is known about the biological function of SCYL3. Here, we show that the biochemical and cell biological properties of SCYL3 are similar to those of SCYL1 and both proteins work in conjunction to maintain motor neuron viability. Specifically, although lack ofScyl3in mice has no apparent effect on embryogenesis and postnatal life, it accelerates the onset of the motor neuron disorder caused byScyl1deficiency. Growth abnormalities, motor dysfunction, hindlimb paralysis, muscle wasting, neurogenic atrophy, motor neuron degeneration, and loss of large-caliber axons in peripheral nerves occurred at an earlier age inScyl1/Scyl3double-deficient mice than inScyl1-deficient mice. Disease onset also correlated with the mislocalization of TDP-43 in spinal motor neurons, suggesting that SCYL1 and SCYL3 regulate TDP-43 proteostasis. Together, our results demonstrate an overlapping role for SCYL1 and SCYL3in vivoand highlight the importance the SCYL family of proteins in regulating neuronal function and survival. Only male mice were used in this study.SIGNIFICANCE STATEMENTSCYL1 and SCYL2, members of the SCY1-like family of pseudokinases, have well established roles in neuronal function. Herein, we uncover the role of SCYL3 in maintaining motor neuron viability. Although targeted disruption ofScyl3in mice had little or no effect on embryonic development and postnatal life, it accelerated disease onset associated with the loss ofScyl1, a novel motor neuron disease gene in humans.Scyl1andScyl3double-deficient mice had neuronal defects characteristic of amyotrophic lateral sclerosis, including TDP-43 pathology, at an earlier age than didScyl1-deficient mice. Thus, we show that SCYL1 and SCYL3 play overlapping roles in maintaining motor neuronal viabilityin vivoand confirm that SCYL family members are critical regulators of neuronal function and survival.