Cryptococcus neoformans is an opportunistic fungal pathogen with a propensity to infect the central nervous system of immune compromised individuals causing life-threatening meningoencephalitis. Cryptococcal biofilms have been described as a protective niche against microbial predators in nature and shown to enhance resistance against antifungal agents and specific mediators of host immune responses. Based on the potential importance of cryptococcal biofilms to its survival in the human host and in nature, these studies were designed to investigate those factors that mediate biofilm formation by C. neoformans. We observed that C. neoformans preferentially grew as planktonic cells when cultured under specific conditions designed to mimic growth within host tissues (37°C, neutral pH, and ˜5% CO2) or phagocytes (37°C, acidic pH, and ˜5% CO2) and as biofilms when cultured under conditions such as those encountered in the external environment (25–37°C, neutral pH, and ambient CO2). Altogether, our studies suggest that conditions similar to those observed in its natural habitat may be conducive to biofilm formation by C. neoformans.