Mesenchymal stem cells (MSCs) are multipotent progenitor cells found in bone marrow that have the capacity of differentiating into bone, cartilage, fat, muscle, and other tissues. Chicken MSCs were isolated from 1- to 14-day-old chickens. Microscopically, the cultured cells showed morphology resembling fibroblasts and divided actively. Chicken MSCs expressed the transcription factors PouV, Sox2, and Nanog, which have been shown to be critical for stem cell self-renewal and pluripotency. The multilineage differentiation potential of chicken MSCs was revealed by their ability to undergo adipogenic, osteogenic, and chondrogenic differentiation. Like mammalian MSCs, chicken MSCs also had immunoregulatory activity and inhibited in vitro mitogenic response of T cells. The inhibition of mitogenic response of T cells correlated with the production of nitric oxide (NO) in cultures containing MSCs and T cells. Our data show for the first time that MSCs can be isolated from postnatal chicken bone marrow and these cells are capable of in vitro multiplication and multilineage differentiation, thus making them a suitable model in the field of stem cell research.