<p>Adipose-derived mesenchymal stromal cells (ASCs) are important components of the breast tumor microenvironment (TME) with hemostatic features and immunomodulatory abilities that can either inhibit or promote tumor growth through direct cell-cell contact or the secretion of biomolecules. This study aimed to investigate the effects of ASCs on PD-1 and/or TIM-3 expression by T cells isolated from breast tumor-draining lymph nodes (TDLNs). Lymphocytes were isolated from fresh, uninvolved axillary lymph nodes and cultured either directly with ASCs from the breast adipose tissue of healthy women (N.ASCs) or breast cancer patients (C.ASCs), or in conditioned media (CM) from these ASCs. After 48 and 72&#xa0;h, the expression of PD-1 and/or TIM-3 on CD3⁺, CD4⁺, and CD8⁺ T cells was analyzed using flow cytometry. Results showed that direct co-culture with both N.ASCs and C.ASCs significantly increased the frequency of CD3⁺, CD3⁺CD4⁺, and CD3⁺CD8⁺T cells expressing the exhaustion marker TIM-3 in both 48- and 72-hour cultures. This increase was independent of PD-1 expression, as both TIM-3⁺PD-1⁺ and TIM-3⁺PD-1^– T cell populations increased significantly. Interestingly, the frequency of T cell populations that expressed PD-1 without TIM-3 remained unchanged. Furthermore, culturing lymphocytes in CM of ASCs had no significant impact on the frequency of TIM-3- and/or PD-1-expressing T cell populations. Additionally, the effect of N.ASCs on TIM-3 and PD-1 expression showed no significant difference from that of C.ASCs. In conclusion, ASCs can contribute to T cell differentiation into a terminally exhausted phenotype by significantly increasing the expression of TIM-3 on these cells, primarily through direct contact.</p>