The purpose of our investigations was to measure, in a co-culture condition, the immunoresponse to allogeneic or xenogenic cells, selected as potential sources for cell therapy of arthritis. We challenged human spleen-derived cells (hSpl) by three different mechanisms: 1) exposure to donor allogeneic or xenogeneic cellular antigens; 2) exposure to donor cells transduced with adenoviral antigens (Ad) and 3) lipopolysaccharide (LPS), a known inflammatory immunostimulant. The immunoresponse to allogeneic human synovial-derived mesenchymal stromal cells alone or transduced with adenoviral green fluorescent protein (hSD-MSC or hSD-MSC/GFP) or the immunoresponse to xenogeneic equine mesenchymal stromal cells (eqMSC) or equine dermal fibroblasts (eqDFb), characterized by the proportion of CD3⁺, CD4⁺, and CD8⁺ human splenocytes (hSpl), was measured on Day 0 and Day 6 of co-culture by flow cytometry. In culture with hSD-MSC, hSD-MSC/GFP, eqDFb, or eqMSC, the proportion of CD3⁺ and CD8⁺ hSpl increased with time in culture but not with exposure to cell alloor xeno-antigens. Both hSD-MSC and hSD-MSC/GFP increased in number during culture and were not affected in viability or proliferation by co-culture with allogeneic hSpl. In this in vitro, primary exposure study, hSpl demonstrated a natural selection and adaptation to a short-term cell culture environment, and that neither allogeneic nor xenogeneic cell antigens incited a greater cellular immunoactivation than co-cultured hSpl alone.