The presence of astrocyte precursor cells (APCs) and time course and topography of astrocyte differentiation during development were investigated by triple‐label immunohistochemistry with intact fetal and adult human retinas. Throughout retinal development and adulthood, expression of Pax2 was restricted to cells of the astrocytic lineage. Three distinct stages of astrocytic differentiation were identified during development: i) Pax2⁺/vimentin⁺/GFAP^‐ APCs; ii) Pax2⁺/vimentin⁺/GFAP⁺ immature perinatal astrocytes; and iii) Pax2⁺/vimentin^‐/GFAP+ mature perinatal astrocytes. In adult, cells with the antigenic phenotype of mature perinatal astrocytes were restricted to a region surrounding the optic nerve head (ONH), whereas cells at a fourth stage of differentiation, adult astrocytes (Pax2^‐/vimentin^‐/GFAP⁺), were apparent throughout the vascularized retina. APC appearance was centered around the ONH and preceded the appearance of perinatal astrocytes. A cluster of Pax2⁺ somas was also present in a small region surrounding the ONH at the ventricular surface of the developing retina, which suggests the existence of two distinct sites of astrocytic differentiation. The coincidence in the location of APCs and perinatal astrocytes at the ventricular zone with that of optic nerve colobomas, together with the association of Pax2 gene mutations with this condition, suggests that coloboma formation may result from impaired astrocyte differentiation during development.