Abstract: Based on the tissue culture,the effect of N-phenyl-N’-1,2,3-thiadiazol-5-ylurea(TDZ),6-benzyla minoprine(6-BA) and 1-Naphthylacetic acid(NAA) on spore germination,protonema development and bud differentiation in Entodon challengeri was investigated in this paper.The whole process was observed and taken pictures.The results show that TDZ,6-BA and NAA have no significant effect on the spore germination.During the process of protonema development,1.0 mg/L NAA can promote the primary development of protonema significantly,and 0.4 mg/L TDZ promotes branches formation in intermediate stage of the protonema development.The number of bud increases with the concentrations augmentation of the three growth regulators,but it is better under 0.4 mg/L TDZ.And it is the best effect when 1.5 mg/L 6-BA and TDZ is mixed.The bud quantity isn’t correlated directly with its growing trend.