Several nonvertebrate hosts has been used to investigate virulence traits and therapies for several bacterial and fungal pathogens. This chapter details the use of heterologous hosts for the study of Cryptococcus neoformans. A striking finding was the correlation between known mammalian virulence factors such as the capsule, melanin, and phospholipase and their necessity for survival in amoebae. Many of the assays used when working with macrophages can be modified for use with Acanthamoeba castellanii, including killing assays, trypan blue staining, and phagocytosis assays. Although many virulence traits have shown some concordance in their requirement for both mammalian and protozoan virulence, there are some discrepancies. Two assays have been developed using Caenorhabditis elegans as a host to study C. neoformans. The first assay, termed the “killing assay”, was initially utilized to identify C. neoformans mutants reduced in virulence. Another assay has been developed based on the observation of smaller nematode brood size when infected with pathogenic C. neoformans. Mutants of Pka1 or Gpa1 (the α subunit) are reduced in virulence in C. elegans, similar to the observed virulence in mice studies. Acapsular mutants also displayed attenuated virulence in Dictyostelium discoideum and Galleria mellonella infection assays, although they remain virulent in D. melanogaster infection assays. Since the Cryptococcus genus includes a large set of organisms, all of which are also presumably under ameboid predator selection in soils and are able to survive in their environments, the avirulence of most species represents the absence of a complete trait set for animal hosts.