Embryonic stem cells (ESC) hold tremendous potential for therapeutic applications, including regenerative medicine, as well as for understanding basic mechanisms in stem cell biology. Since numerous experiments cannot be conducted in human ESC because of ethical or practical limitations, nonhuman primate ESC serve as invaluable clinically relevant models. The novel marmoset (Callithrix jacchus) ESC line cjes001 was characterized using different stem cell markers. The cells were stained positively with Oct4, SSEA-3, SSEA-4, Tra-1-60, Tra-1-81, and Sox-2 underscoring their status as undifferentiated ESC. ESC are typically grown on mouse embryonic fibroblasts (MEF) as feeder cells whose proliferation is arrested either by treatment with Mitomycin C or by gamma-irradiation. To assess the impact of these treatments on the ability of MEF to support the growth of undifferentiated ESC, we used an MTT assay to evaluate the cellular metabolic activity of growth arrested feeder cells. There was a significant (p < 0.02) difference in gamma-irradiated cells displaying a higher metabolic activity compared to Mitomycin C inactivation. Also we quantified 69 soluble factors in the supernatant of both Mitomycin-treated and gamma-irradiated MEF by bead-based multiplex analysis, and thus established a profile of MEF-secreted factors. The time course of secretion was analyzed by monitoring the supernatant at 0, 6, 12, and 24 h after changing the medium. Comparing gamma-irradiated and Mitomycin-treated MEF suggested higher amounts of some cytokines including FGF or SCF by the former. We also assessed whether the method of inactivation had an effect on growth kinetics and differentiation of primate ESC. There appeared to be a trend to a lower number of differentiated ESC colonies on the gamma-irradiated feeder cells, suggesting that this may be the preferable method of growth arrest.