Acquired transplant tolerance could be readily induced during foetal or neonatal period through donor cell infusion, but it is not the case in adults. This phenomenon has been attributed to the variation of immune system development in neonatal and adult periods. To investigate the role of immature immune system and chimerism in neonatal transplant tolerance, irradiated spleen cells or cell fraction from F1 (BALB/c × C57BL/6) or GFP‐F1 mice were injected intravenously into neonatal C57BL/6 mice to induce tolerance. Irradiated cells or cell fraction could not induce chimerism and transplant tolerance in neonatal mice, even increasing the dose of donor cells to 5 × 107. Living donor cells induced tolerance in neonatal mice, and the quantity of living cells was correlated with the degree of chimerism and tolerance. At the amount of 3 × 107 F1 spleen cells, skin grafts were survived permanently in more than 80% of treated mice. However, the amount of 0.7 × 107 F1 spleen cells could only slightly prolong allografts’ survival. The more donor cells were infused, the higher level of chimerism was achieved, and the higher frequency of alloreactive T cells was deleted. Chimerism is prerequisite for the induction and maintenance of tolerance. Chimerism in long‐term tolerant mice was significantly higher than that in chronic graft rejected mice, with 6.48 ± 4.02% versus 1.41 ± 0.77%. It implies that transplant tolerance depends on the establishment of chimerism, but not on antigen exposure to immature immune system in foetus or neonates.