We recently demonstrated that a goldfish macrophage cell line (GMCL) and primary in vitro-derived kidney macrophage (IVDKM) cultures contain three distinct macrophage subpopulations. Morphological, cytochemical, functional, and flow cytometric characterization of these sub-populations suggested that they may represent cells of the macrophage lineage temporally arrested at distinct differentiation junctures of fish macrophage development (putative early progenitors, monocytes, and macrophages). In this study, we examined the proliferation and differentiation events leading to the generation of mature macrophage-like cells from goldfish kidney hematopoietic tissues. The flow cytometric studies were done after labeling macrophages with PKH26 fluorescent dye and analysis of the data using the MODFIT software. Our results showed that IVDKM cultures proliferated non-synchronously, suggesting the presence of a temporal control mechanism regulating the number of cells entering the paths towards maturation. Such control is most evident during early progenitor proliferation and differentiation events. Our results showed that proliferation may not be a requirement for differentiation of early progenitors to putative monocyte and macrophage subsets. Detailed observation of the mature macrophage-like subpopulation indicated that: 1) they appear to develop from both, the differentiation of monocyte-like cells, and direct differentiation of early progenitors in the absence of a monocyte-like stage; and (2) mature macrophage-like cells appeared to be capable of self-proliferation. Our results suggest the presence of alternate pathways of fish macrophage development other than the classical hematopoietic pathway.