Pre-mRNA (messenger RNA) splicing participates in the regulation of numerous biological processes in plants. For example, alternative splicing shapes transcriptomic responses to abiotic and biotic stress, and controls developmental programs. However, no study has revealed a role for splicing in maintaining the root stem cell niche. Here, a screen for defects in root growth inArabidopsis thalianaidentified an ethyl methane sulfonate mutant defective in pre-mRNA splicing (rdm16-4). Therdm16-4mutant displays a short-root phenotype resulting from fewer cells in the root apical meristem. ThePLETHORA1(PLT1) andPLT2transcription factor genes are important for root development and were alternatively spliced inrdm16-4mutants, resulting in a disordered root stem cell niche and retarded root growth. The root cap ofrdm16-4contained reduced levels of cytokinins, which promote differentiation in the developing root. This reduction was associated with the alternative splicing of genes encoding cytokinin signaling factors, such asARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN5andARABIDOPSIS RESPONSE REGULATORS(ARR1, ARR2, andARR11). Furthermore, expression of the full-length coding sequence ofARR1or exogenous cytokinin application partially rescued the short-root phenotype ofrdm16-4. This reveals that the RDM16-mediated alternative splicing of cytokinin signaling components contributes to root growth.