The human respiratory pathogen Haemophilus influenzae, a Gram-negative bacterium, is the first free-living organism to have its complete genome sequenced, providing the opportunity to apply genomic-scale approaches to study gene function. This chapter provides an overview of a highly efficient, in vitro mariner transposon-based method that exploits the natural transformation feature of this organism for the identification of essential genes. In addition, we describe strategies for conditional expression systems that would facilitate further analysis of this class of genes. Finally, we outline a method based on the approach used in H. influenzae for identifying essential genes that can be applied to other bacteria that are not naturally transformable.