Growth factors and cytokines including FLT3 Ligand (FLT3L) have many applications in cellular and molecular biology studies, and also as therapeutical agents. FLT3L was expressed in Pichia pastoris through cloning from human leukemic K562 cell line as well as using a codon-optimized synthetic construct. Codon adaptation index (CAI) was increased from 55 in the native sequence up to 95 after optimization. Significant changes occurred in the codons for Pro, Arg, Leu and Ser toward the favored codons in P. pastoris. Both forms of expressed FLT3L (from the native and optimized sequences) were capable of stimulating proliferation of the FDC-P1 cells expressing human wild-type FLT3 (FD–FLT3–WT). Sequence optimization resulted in 55-fold increase in the yield of active FLT3L (290 μg/mL of product in the crude supernatant). Amino acid residues 27–162 are sufficient for the biological function of human FLT3L. Pichia pastoris is a highly efficient and cost-effective system for expression of endotoxin-free FLT3L; however, codon optimization is necessary for its optimal expression.