Multiple shoots were produced directly from each explant. BA, zeatin, or TDZ were evaluated on callus initiation, development, and shoot organogenesis. Callus production was promoted when BA or zeatin was added in culture medium. However, no shoots were produced from such calli. Multiple shoots were produced directly through shoot organogenesis from each seed explant when TDZ was added to culture medium. As many as 30 to 40 shoots were produced per seed explant in about 7 weeks from culture initiation. Different alfalfa cultivars were also tested. The established multiple shoot production protocol provides an efficient way to produce transgenic alfalfa plants, thus could significantly advance alfalfa genetic transformation.