Wnt5a can activate Rho GTPases in chronic lymphocytic leukemia (CLL) cells by inducing the recruitment of ARHGEF2 to ROR1. Mass spectrometry on immune precipitates of Wnt5a-activated ROR1 identified 14–3–3ζ, which was confirmed by co-immunoprecipitation. The capacity of Wnt5a to induce ROR1 to complex with 14–3–3ζ could be blocked in CLL cells by treatment with cirmtuzumab, a humanized mAb targeting ROR1. Silencing 14–3–3ζ via small interfering RNA impaired the capacity of Wnt5a to: (1) induce recruitment of ARHGEF2 to ROR1, (2) enhance in vitro exchange activity of ARHGEF2 and (3) induce activation of RhoA and Rac1 in CLL cells. Furthermore, CRISPR/Cas9 deletion of 14–3–3ζ in ROR1-negative CLL cell-line MEC1, and in MEC1 cells transfected to express ROR1 (MEC1-ROR1), demonstrated that 14–3–3ζ was necessary for the growth/engraftment advantage of MEC1-ROR1 over MEC1 cells. We identified a binding motif (RSPS857SAS) in ROR1 for 14–3–3ζ. Site-directed mutagenesis of ROR1 demonstrated that serine-857 was required for the recruitment of 14–3–3ζ and ARHGEF2 to ROR1, and activation of RhoA and Rac1. Collectively, this study reveals that 14–3–3ζ plays a critical role in Wnt5a/ROR1 signaling, leading to enhanced CLL migration and proliferation.