Gene therapy based on the use of viral vectors is entirely dependent on the use of animal cell lines, mainly of mammalian origin, but also of insect origin. As for any biotechnology product for clinical use, viral -vectors have to be produced with cells derived from an extensively characterized cell bank to maintain the appropriate standard for assuring the lowest risk for the patients to be treated. Although many different cell types and lines have been used for the production of viral vectors, HEK293 cells or their derivatives have been extensively used for production of different vector types: adenovirus, oncorectrovirus, lentivirus, and AAV vectors, because of their easy handling and the possibility to grow them adherently in serum-containing medium as well as in suspension in serum-free culture medium. Despite this, these cells are not necessarily the best for the production of a given viral vector, and there are many other cell lines with significant advantages including superior growth and/or production characteristics, which have been tested and also used for the production of clinical vector batches. This chapter presents basic -considerations concerning the characterization of cell banks, in the first part, and, in the second part, practically all cell lines (at least when public information was available) established and developed for the production of the most important viral vectors (adenoviral, oncoretroviral, lentiviral, AAV, baculovirus).