Meningiomas are common central nervous system tumors; however, the mechanisms underlying their pathogenesis are largely unknown. Collaborative studies from our laboratory demonstrated a direct association of 14-3-3 with the meningioma tumor suppressor Protein 4.1B, which was not observed with other members of the Protein 4.1 family, including the NF2 meningioma tumor suppressor, merlin/schwannomin. Given the role of 14-3-3 in the regulation of cell proliferation and apoptosis, we sought to determine the functional significance of 14-3-3 binding to Protein 4.1B growth suppression. Based on comparative binding studies performed with additional members of the Protein 4.1 family, we generated specific missense mutations within the minimal growth suppressor fragment of Protein 4.1B (DAL-1, differentially expressed in adenocarcinoma of the lung). Complementary in vitro GST affinity chromatography and in vivo interaction experiments demonstrated that the F359Y mutation abrogated binding to 14-3-3, but did not impair DAL-1 binding to other known Protein 4.1B interacting proteins. Similar to wild-type DAL-1, the expression of the F359Y DAL-1 14-3-3-binding mutant resulted in reduced Protein 4.1B-deficient IOMM-Lee and CH157-MN meningioma cell line colony formation. Moreover, similar to wild-type DAL-1, the stable expression of the DAL-1 F359Y mutant significantly reduced cell proliferation in independently isolated IOMM-Lee clones, as assessed by thymidine incorporation. Collectively, these results suggest that binding to 14-3-3 is not essential for the growth suppressor function of Protein 4.1B in meningiomas.