An 8-month-old patient was found to have an adrenal cortical carcinoma (ACC) characterized by a TP53 c.758C>T (p.T253I) mutation in the TP53 tumor suppressor gene. p53 protein levels were overexpressed in the patient’s ACC, and molecular profiling of the tumor suggested suppression of the remaining wild-type (WT) TP53 allele. As this type of malignancy has been reported to occur in Li-Fraumeni Syndrome (LFS) patients with germline inheritance of loss-of-function TP53 mutations, we evaluated the patient’s TP53 gene in non-tumor tissue and found evidence of a constitutional heterozygous germline TP53 c.758C>T (p.T253I) variant. Since the mutation has not yet been linked to LFS, we sought to characterize the functionality of the T253I mutation. We acquired p53 ^-/- HEK293 cells and stably transduced them with GFP-tagged wild type (T253) or T253I p53 as well as two established pathogenic p53 mutants (C176Y and R213X). Compared to p53 WT, levels of T253I p53 increased while MDM2 levels decreased, suggesting a loss of MDM2-mediated regulation of T253I p53. Additionally, T253I showed a reduction in DNA damage responsive events, diminished DNA binding capabilities, and blunted transactivation capacity. These experimental data, coupled with clinical observations, lead us to conclude that T253I represents a pathologic variant in TP53 that may predispose to LFS-associated tumors.