Receptors localized at the plasma membrane are critical for the recognition of pathogens. The molecular determinants that regulate receptor transport to the plasma membrane are poorly understood. In a screen for proteins that interact with the FLAGELIN-SENSITIVE2 (FLS2) receptor using Arabidopsis thaliana protein microarrays, we identified the reticulon-like protein RTNLB1. We showed that FLS2 interacts in vivo with both RTNLB1 and its homolog RTNLB2 and that a Ser-rich region in the N-terminal tail of RTNLB1 is critical for the interaction with FLS2. Transgenic plants that lack RTNLB1 and RTNLB2 (rtnlb1 rtnlb2) or overexpress RTNLB1 (RTNLB1ox) exhibit reduced activation of FLS2-dependent signaling and increased susceptibility to pathogens. In both rtnlb1 rtnlb2 and RTNLB1ox, FLS2 accumulation at the plasma membrane was significantly affected compared with the wild type. Transient overexpression of RTNLB1 led to FLS2 retention in the endoplasmic reticulum (ER) and affected FLS2 glycosylation but not FLS2 stability. Removal of the critical N-terminal Ser-rich region or either of the two Tyr-dependent sorting motifs from RTNLB1 causes partial reversion of the negative effects of excess RTNLB1 on FLS2 transport out of the ER and accumulation at the membrane. The results are consistent with a model whereby RTNLB1 and RTNLB2 regulate the transport of newly synthesized FLS2 to the plasma membrane.