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Summary The ability to genetically manipulate organisms has led to significant insights into functional genomics in many species. In birds, manipulation of the genome is hindered by the inaccessibility of the one-cell embryo. During embryonic development, avian primordial germ cells (PGCs) migrate through the bloodstream and reach the gonadal anlage, where they develop into mature germ cells. Here, we explored the use of PGCs to produce transgenic offspring in the zebra finch, which is a major animal model for sexual brain differentiation, vocal learning, and vocal communication. Zebra finch PGCs (zfPGCs) obtained from embryonic blood significantly proliferated when cultured in an optimized culture medium and conserved the expression of germ and stem cell markers. Transduction of cultured zfPGCs with lentiviral vectors was highly efficient, leading to strong expression of the enhanced green fluorescent protein. Transduced zfPGCs were injected into the host embryo and transgenic songbirds were successfully generated., Graphical abstract, Highlights • Blood-derived zebra finch primordial germ cells (zfPGCs) can be expanded in vitro • Lentiviral vectors can efficiently transduce zfPGCs in vitro • Genetically modified zfPGCs can be used to generate transgenic zebra finches, In this article, Gessara and colleagues used zebra finch primordial germ cells obtained from embryonic blood for the genetic modification of a songbird species using lentiviral vectors.