We report here for the first time callus formation from protoplasts in date palm (Phoenix dactylifera L.). Protoplasts were isolated from young leaves of offshoots and embryogenic calli in Deglet nour and Takerboucht genotypes. The protoplast yield depended on genotype, donor plant material, mixture of enzyme solution, and incubation time. With regard to the donor material, the best response was obtained with callus. Cell division was induced in both liquid culture and nurse culture. The best donor material for cell division was callus and the best response was obtained with the feeder layer, which induced a division rate of 30% in Deglet nour and 15% in Takerboucht genotypes. The dividing cells developed to microcalli on the feeder layer; the microcalli developed to calli on modified MS medium; however, the calli failed to regenerate into roots or shoots.