Different hormones, length of hormone pretreatment, and developmental status of plants were found to influence the shoot and root differentiation frequencies from immature inflorescence explants of Medicago lupulina L., a diploid autogamous species. Explants of immature inflorescence pretreated with phytohormones for a short period (10 min −6 h) were placed on hormone-free medium for shoot differentiation. Cytokinins, such as BA, were found to be essential for shoot differentiation, whereas treatment with 2,4-D could only induce root differentiation. Explants with hormone pretreatment for 30 min to 1 h produced the highest shoot regeneration rate. Plants at the beginning of flowering were found to have a better regeneration ability than those at fruiting or maturation. After a 10 mg/l BA pretreatment, respective shoot differentiation frequencies of 39%, 36% and 23% were observed for explants of self-progeny plants, regenerated plants, and plants propagated by cuttings, although the three categories of plants were originally derived from the same plants.