Termite soldiers represent a peculiar caste among social insects in terms of their specific defensive roles. Numbers of soldiers are relatively low in a mature colony, and it is impossible to identify the individuals that will differentiate into soldiers. If it were possible to specify these individuals prior to soldier differentiation, it would facilitate a better understanding of the regulatory mechanisms of soldier differentiation under natural condition. Here we analyzed soldier differentiation in incipient colonies of Zootermopsis nevadensis, in which only a single soldier develops via a presoldier stage, and is stable during early colony ontogeny. We observed that the oldest third instar differentiated into a presoldier within about eight days from its appearance. Caste differentiation, however, was not strictly determined on an individual basis. The oldest third instars never differentiated into presoldiers if primary reproductives were removed soon after their appearance. Behavioral observations of primary reproductives and their offspring prior to presoldier differentiation, showed that primary reproductives transferred proctodeal materials to the oldest third instar significantly more frequently than to other larva. A high juvenile hormone (JH) titer is required for the soldier differentiation, and we suggest that the JH itself or some nutrients/factors increasing larval JH titer may be transferred to the oldest third instar via a parental proctodeal fluid.