<p>Immune checkpoints expressed by immune cells are critical mediators of immune suppression in cancer. Recently, the role of VISTA, an immune checkpoint, in suppressing T cells has been highlighted in several studies. However, its involvement in B-cell acute lymphoblastic leukemia (B-ALL) remains underexplored. In this study, we analyzed the expression of VISTA, the immune-regulatory marker CD244, and its corresponding ligand CD48. Additionally, we examined the expression of the transcription factors FOXD3 and PVRL2 in pediatric patients with B-ALL. Peripheral blood samples from pediatric patients with naïve B-ALL were analyzed using flow cytometry. Additionally, real-time PCR was used to evaluate the downstream regulatory gene FOXD3 expression and the immune regulator PVRL2. VISTA was overexpressed on blasts in B-ALL patients. Also, the frequency of CD3⁺CD8⁺VISTA⁺ T cytotoxic cells was significantly higher in patients compared to controls, while CD3⁺CD4⁺VISTA⁺ T helper cells were reduced. CD19⁺VISTA⁺ cells were more abundant in the complete remission group compared to the non-complete remission group. FOXD3, a key regulator of VISTA, was significantly downregulated in B-ALL, consistent with VISTA overexpression. The CD244/CD48 interaction, which can promote anti-tumoral immune responses, showed a reduction in CD3⁺CD4⁺CD48⁺ and CD19⁺CD48⁺ cells, while CD3⁺CD8⁺CD48⁺ cells were increased. VISTA overexpression and FOXD3 downregulation in B-ALL, alongside altered CD48, and PVRL2 expression, highlight mechanisms of immune evasion. These findings position VISTA as a promising biomarker and target for B-ALL immunotherapy.</p>